Peptide inhibitors of a phosphotyrosine-binding domain containing protein

ABSTRACT

A peptide of the formula (I): X 1  -A 1  -A 2  -X 2  -Asn-X 3  -X 4  -P.Tyr-X 5  -X 6  -X 7  -X 8 , wherein X 1  represents Lys, Arg, His, Ser, Thr, Tyr, Asn, Leu, Val or Glu, A 1  represents Trp, Leu, Ala, Ser, Ile, Glu, Met, Gly, Cys, Phe, Pro or Val, and A 2  represents Ala, Val, Leu, Ile, Ser, Met, Phe, Gly, Cys, Trp or Pro, X 2  represents Glu, Asn, Tyr, Thr, Ser, Asp or Ile, X 3  represents Pro, Met, Trp, Phe, Ala, Lys, Val, Leu, Ile, Gly or Ser, X 6  represents Ser, Thr, Tyr, Asn, Glu, Met, Ala, Leu, Val or Gly, X 7  represents Asp, Glu, Ala, Val, Leu, Ile, Gly, Cys, Phe, Trp, Met, Pro, Ser or Asn and X 8  which may be present or absent represents Phe, Trp, Pro, Leu, Ala, Val, Ile, Gly, Cys, Met, Asp, Ser or Arg which interferes with the interaction of a PTB domain containing protein with a PTB domain binding site, and truncations and analogues of the peptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US96/17080, filed Oct. 24, 1996, whichclaims the benefit under Title 35, United States Code § 119(e) ofProvisional application Ser. Nos. 60/005,944, 60/010,384, and 60/011,799filed Oct. 27, 1995, Jan. 22, 1996, and Feb. 20, 1996, respectively.

FIELD OF THE INVENTION

The invention relates to peptides which interfere with the interactionof a phosphotyrosine-binding (PTB) domain containing protein with a PTBdomain binding site; and, uses of the peptides.

BACKGROUND OF THE INVENTION

Shc is a member of a group of proteins that are collectively known asadaptor proteins. These adaptors, which are composed of protein-proteininteraction domains such as the Src-homology 2 (SH2) and Src-homology 3(SH3) domains, mediate protein-protein interactions that are importantfor signal transduction downstream of growth factor and cytokinereceptors (Pawson, 1995). Shc has been shown to bind to a wide varietyof activated growth factor and cytokine receptors. Shc was cloned from ahuman cDNA library in a screen for SH2 domain-containing proteins(Pelicci et al., 1992); Shc homologs in mouse (mShc) and drosophila(dShc) have also been cloned (Lai et al., 1995). Three proteins areencoded by the shc gene that differ from each other only in theiramino-terminus (Lai et al., 1995; Pelicci et al., 1992). Overexpressionof Shc results in cellular transformation of NIH3T3 fibroblasts andRas-dependent neurite outgrowth of PC12 cells, suggesting that Shc playsan important role in signal transduction leading to DNA synthesis andcell division or differentiation (Pelicci et al., 1992; Rozakis-Adcocket al., 1992).

Shc contains an amino-terminal phosphotyrosine-binding (PTB) domain, acentral Pro-rich region that contains the principal tyrosinephosphorylation site at Tyr 317, and an SH2 domain at itscarboxy-terminus. The PTB domain, which is highly conserved inShc-related proteins, was recently identified based on its ability tobind to phosphotyrosine-containing proteins (Blaikie et al., 1994;Kavanaugh and Williams, 1994; van der Geer et al., 1995). It recognizesphosphotyrosine present within the sequence Asn-Pro-X-P.Tyr and differsfrom SH2 domains that recognize phosphotyrosine in the context ofcarboxy-terminal residues (Kavanaugh et al., 1995; van der Geer et al.,1995). The Shc SH2 domain recognizes phosphotyrosine within the sequenceP.Tyr-Glu/Leu/Ile/Tyr-X-Leu/Ile/Met (Songyang et al., 1994).

Shc becomes phosphorylated on tyrosine following stimulation with a widevariety of growth factors and cytokines (Burns et al., 1993; Crowe etal., 1994; Cutler et al., 1993; Lanfrancone et al., 1995; Pelicci etal., 1992; Pronk et al., 1993; Ravichandran et al., 1993; Segatto etal., 1993; Yokote et al., 1994). Tyrosine phosphorylation of Shc isessential for its interaction with the Grb2-Sos complex, which mayprovide a mechanism for Ras activation (Buday and Downward, 1993; Croweet al., 1994; Egan et al., 1993; Gale et al., 1993; Li et al., 1993;Rozakis-Adcock et al., 1993; Rozakis-Adcock et al., 1992; Salcini etal., 1994). Shc has also been shown to bind physically to activatedgrowth factor and cytokine receptors. Several growth factor receptorsthat had previously been shown to bind to Shc upon activation containtyrosine phosphorylation sites present within the sequenceAsn-Pro-X-P.Tyr, consistent with the notion that it is the PTB domainthat mediates Shc's interaction with these proteins (Campbell et al.,1994; van der Geer and Pawson, 1995). Furthermore, the Shc PTB domainhas been shown to bind to the activated nerve growth factor (NGF)receptor, the activated epidermal growth factor (EGF) receptor, polyomamiddle T antigen, and to a 145 kDa protein that becomes phosphorylatedon tyrosine in PDGF stimulated cells (Blaikie et al., 1994; Kavanaughand Williams, 1994; van der Geer et al., 1995). The NGF receptorcontains a single Shc-binding site at Tyr 490 that is present within aAsn-Pro-X-Tyr motif (Obermeier et al., 1994; Stephens et al., 1994). NGFreceptors that have been mutated at Tyr 490 lack the ability to interactwith Shc in vivo or with the PTB domain in vitro (Stephens et al.,1994). Phosphotyrosine-containing peptides based on the Shc binding sitein middle T antigen, which is also present within an Asn-Pro-X-P.Tyrmotif, compete with the NGF and EGF receptors for binding to the PTBdomain (van der Geer et al., 1995).

SUMMARY OF THE INVENTION

The present inventors have identified the residues within theAsn-Pro-X-P.Tyr motif of phosphotyrosine-containing proteins (e.g.growth activated growth factors and cytokine receptors) that mediate thebinding of the proteins to signalling proteins containing PTB domains.In particular, the present inventors found that the Asn and thephosphotyrosine residues within the Asn-Pro-X-P.Tyr motif of thephosphotyrosine-containing proteins mediate their binding to the PTBdomain of Shc. The present inventors also found that an aliphaticresidue that is five or six residues amino-terminal to thephosphotyrosine is required for binding. This aliphatic residue ismissing from the insulin receptor autophosphorylation site which isunable to form a stable complex with Shc. The present inventors alsoanalyzed the Shc PTB domain by in vitro mutagenesis and anevolutionarily conserved Arg residue was identified that is importantfor PTB binding to its ligands.

Broadly stated the present invention relates to a peptide of the formulaI

    X.sup.1 -A.sup.1 -A.sup.2 -X.sup.2 -Asn-X.sup.3 -X.sup.4 -P.Tyr-X.sup.5 -X.sup.6 -X.sup.7 -X.sup.8                                I

wherein X¹ represents Lys, Arg, His, Ser, Thr, Tyr, Asn, Leu, Val, orGlu, A¹ represents Trp, Leu, Ala, Ser, Ile, Glu, Met, Gly, Cys, Phe,Pro, or Val, and A² represents Ala, Val, Leu, Ile, Ser, Met, Phe, Gly,Cys, Trp, or Pro, X² represents Glu, Asn, Tyr, Thr, Ser, Asp, or Ile, X³represents Pro, Met, Trp, Phe, Ala, Lys, Val, Leu, Ile, Gly, or Cys, X⁴represents Leu, Ala, Glu, Gin, Asp, Asn, Tyr, Thr, or Ser, X⁵ representsPhe, Trp, Pro, Leu, Ala, Val, Ile, Gly, Cys, Met, Arg or Ser, X⁶represents Ser, Thr, Tyr, Asn, Glu, Met, Ala, Leu, Val, or Gly, X⁷represents Asp, Glu, Ala, Val, Leu, Ile, Gly, Cys, Phe, Trp, Met, Pro,Ser, or Asn, and X⁸ which may be present or absent represents Phe, Trp,Pro, Leu, Ala, Val, Ile, Gly, Cys, Met, Asp, Ser, or Arg, whichinterferes with the interaction of a PTB domain containing protein witha PTB domain binding site.

In an embodiment of the present invention a peptide of the formula I isprovided:

    X.sup.1 -A.sup.1 -A.sup.2 -X.sup.2 -Asn-X.sup.3 -X.sup.4 -P.Tyr-X.sup.5 -X.sup.6 -X.sup.7 -X.sup.8                                I

wherein X¹ represents His, Ser, Thr, Tyr, Asn, Leu, Val, or Glu, X²represents Glu, Ser, Asp, or Ile, X³ represents Pro or Lys, X⁴represents Leu, Ala, Glu, Gln, Asn, or Thr, X⁵ represents Phe, Leu, Ile,Gly, Arg, or Ser, X⁶ represents Ser, Thr, Met, Ala, Leu, Val, or Gly, X⁷represents Asp, Ala, Val, Leu, Met, Ser, or Asn, X⁸ which may be presentor absent, represents Leu, Ala, Gly, Asp, Ser, or Arg, A¹ representsTrp, Leu, Ala, Ser, Ile, Glu, Met, or Val, and A² represents Ala, Val,Leu, Ile, Ser, Met, or Phe, which interferes with the interaction of aPTB domain containing protein with a PTB domain binding site.

In another embodiment of the invention a peptide of the formula Ia isprovided

    X.sup.1 -A.sup.1 -A.sup.2 -X.sup.2 -Asn-X.sup.3 -X.sup.4 -P.Tyr-X.sup.5 -X.sup.6 -X.sup.7                                         Ia

wherein X¹ represents Lys, Arg, His, preferably His, X² represents Glu,Asn, Tyr, Thr, Ser, preferably Glu, X³, represents Pro, Met, Trp, Phe,Ala, Val, Leu, Ile, Gly, Cys, preferably Pro, X⁴ represents Gln, Asp,Asn, Tyr, Thr, Ser, preferably Gln, X⁵ represents Phe, Trp, Pro, Leu,Ala, Val, Ile, Gly, Cys, Met, preferably Phe, X⁶ represents Ser, Thr,Tyr, Asn, Glu, preferably Ser, X⁷ represents Asp, Glu, preferably Asp,and one of A¹ and A² represents Ile and the other of A¹ and A²represents Ile or Ala, preferably A¹ represents Ala and A² representsIle, which interferes with the interaction of a PTB domain containingprotein with a PTB domain binding site.

In still another embodiment of the invention a peptide of the formula Iais provided

    X.sup.1 -A.sup.1 -A.sup.2 -X.sup.2 -Asn-X.sup.3 -X.sup.4 -P.Tyr-X.sup.5 -X.sup.6 -X.sup.7                                         Ia

wherein X¹ represents Ser, Thr, Tyr, Asn or Glu, preferably Tyr, X²represents Glu, Asn, Tyr, Thr, Ser, preferably Ser, X³ represents Pro,Met, Trp, Phe, Ala, Val, Leu, Ile, Gly, Cys, preferably Pro, X⁴represents Glu, Asp, preferably Glu, X⁵ represents Phe, Trp, Pro, Leu,Ala, Val, Ile, Gly, Cys, Met preferably Leu, X⁶ represents Ser, Thr,Tyr, Asn, Glu, preferably Ser, X⁷ represents Ala, Val, Leu, Ile, Gly,Cys, Phe, Trp, Met, Pro, preferably Ala, and one of A¹ and A² representsIle and the other of A¹ and A² represents Ala, Val, Leu, Ile, Gly, Cys,Phe, Trp, Met or Pro, which interferes with the interaction of a PTBdomain containing protein with a PTB domain binding site.

The invention also relates to truncations and analogs of the peptides ofthe invention.

The invention also relates to the use of a peptide of the formula I orIa to interfere with the interaction of a PTB domain containing proteinwith a PTB domain binding site; and, pharmaceutical compositions forinhibiting the interaction of a PTB domain containing protein with a PTBdomain binding site.

Further, the invention relates to a method of modulating the interactionof a PTB domain containing protein with a PTB domain binding sitecomprising changing the amino acid Arg at position 175 in the PTB domaincontaining protein. The invention still further relates to a method formodulating the interaction of an insulin receptor with insulin receptorsubstrate 1 (IRS-1) or Shc comprising incorporating a large aliphaticamino acid at amino acids -5 or -6 amino terminal to the P.Tyr in themotif Asn-Pro-X-P.Tyr in the PTB domain of the insulin receptor.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the drawingsin which:

FIG. 1A is an immunoblot showing P.Tyr-containing proteins bound to GST(lane 2) and GST Shc PTB (lanes 1, 3-6) fusion proteins immobilized onglutathione-agarose after incubation with lysates from control (lane 1)and NGF-stimulated (lanes 2-7) cells in the absence (lane 1-3) andpresence of Wt (lane 4) and mutant (lanes 5 and 6) competing P.Tyrcontaining peptides, based on the sequence around Tyr 490 the Shc PTBdomain binding site in the NGF receptor;

FIG. 1B is the immunoblot shown in FIG. 1A stripped and reprobed with anantiserum raised against the NGF receptor;

FIG. 2 is a graph showing the results of surface plasmon resonancetechnology testing the ability of Wt and mutant phosphopeptides, basedon the sequence around Tyr 490 the Shc-binding site in the NGF receptorto compete for binding of the GST-Shc PTB domain fusion protein to theimmobilized polyoma middle T antigen peptide;

FIG. 3 is a schematic diagram showing the presence of an Asn-Pro-X-P.Tyrmotif in the juxta membrane domains of the NGF (SEQ ID NO:56) andinsulin receptors (SEQ ID NO:57);

FIG. 4A is an immunoblot showing anti-Shc immunoprecipitates (lanes 1,2, 5, 6, 9, and 10) from control (lanes 1, 5, and 9) and growthfactor-stimulated (lanes 2, 6, and 10) NIH3T3 fibroblasts expressing Wt(lanes 1 and 2; NGFR) or Phe 490 mutant (lanes 5 and 6; F490NGFR) NGFreceptors, or CHO cells expressing Wt insulin receptors (lanes 9 and 10;IR) analyzed by anti-P.Tyr immunoblotting; anti-NGF receptor (lanes 3,4, 7, and 8) and anti-insulin receptor immuno-precipitates (lanes 11 and12) from control (lanes 3, 7, and 11) and growth factor stimulated(lanes 4, 8, and 12) were analyzed in parallel;

FIG. 4B is an immunoblot showing Wt (lanes 1 and 2) and Phe 490 mutant(5 and 6) NGF receptors present in lysates from control (lanes 1 and 5)and NGF-stimulated (lanes 2 and 6) cells expressing Wt (NGFR) or Phe 490mutant (F490NGFR) and insulin receptors (IR) present in lysates fromcontrol (lane 9) and insulin-stimulated (lane 10) cells incubated withGST-Shc PTB fusion proteins bound to glutathione-agarose, bound proteinswere analyzed by anti-P.Tyr blotting;

FIG. 5A is an immunoblot showing GST-Shc PTB domain fusion proteinsbound to glutathione-agarose after incubation with activated NGFreceptors present in lysates of NGF-stimulated cells in the absence(lane 1) or presence (lanes 2-7) of 2 μM competing Wt and mutantphosphotyrosine containing peptides based on the sequence around Tyr490, the Shc PTB domain binding site in the NGF receptor (lanes 2-5) orTyr 960 an autophosphorylation site present within an Asn-Pro-X-P.Tyrmotif in the insulin receptor (lanes 6 and 7);

FIG. 5B is a graph showing the results of testing phosphopeptides basedon the sequence around Tyr 490, the Shc-binding site in the NGF receptor(H-I-I-E-N-P-Q-p.Y-F-S-D SEQ ID NO:1; () or Tyr. 960 in the insulinreceptor (Y-A-S-S-N-P-E-p.Y-L-S-A SEQ ID NO:30; (o) and substitutions atposition -5 and -6 with respect to the P.Tyr in the NGF receptorpeptides (H-A-S-E-N-P-Q-p.Y-F-S-D (SEQ ID NO:31); (▪)) and the insulinreceptor peptide (Y-A-I-S-N-P-E-p.Y-L-S-A (SEQ ID NO:5); (▴) for theirability to compete for the binding of the GST-Shc PTB domain to theimmobilized polyoma middle T antigen peptide(L-S-L-L-S-N-P-T-p.Y-S-V-M-R-S-K) (SEQ ID NO:23);

FIG. 6A is an immunoblot showing GST fusion proteins containing Wt(lanes 1 and 2) or mutant (lanes 3-11) Shc PTB domains after incubationwith NGF receptors present in lysates of control (lane 1) andNGF-stimulated cells (lanes 2-11), bound proteins were analyzed byanti-P.Tyr blotting;

FIG. 6B is an immunoblot showing human EGF receptors bound to GST fusionproteins containing Wt (lanes 1 and 2) or Met 175 (lane 3) and Lys 175(lane 4) mutant human Shc PTB domains in lysates from control (lane 1)or EGF-stimulated cells (lanes 2-4) analyzed by anti-P.Tyr blotting, andin parallel GST (lane 8) and GST fusion proteins containing Wt (lane 7)or an Ala 151 mutant (lane 9) drosophila Shc PTB domain bound toglutathione-agarose, incubated with fly lysates containing activatedTorso-DER chimeric proteins that contain the cytoplasmic domain of DER;bound proteins were detected by anti-P.Tyr blotting;

FIG. 7 shows the amino acid sequences of PTB binding domains ofmammalian and Drosophila Shc homologues (SEQ ID NOs:58, 59 and 60);

FIG. 8 are immunoblots showing competitive inhibition of EGF receptorbinding to GST-ShcB analyzed by anti-phospho-tyrosine antibody;

FIG. 9 is an immunoblot showing competitive inhibition of EGF receptorbinding to GST-ShcB analyzed by anti-phospho-tyrosine antibody;

FIG. 10 are immunoblots showing a dose-response analysis in acompetitive inhibition assay of EGF receptor binding to GST-ShcBanalyzed by anti-phospho-tyrosine antibody;

FIG. 11a is a bar graph showing proliferation of HER14 cells treatedwith peptides of the invention;

FIG. 11b is a bar graph showing proliferication of HER14 cells treatedwith peptides of the invention;

FIG. 12 is a bar graph showing proliferation of HER14 cells treated withpeptides of the invention;

FIG. 13 is a bar graph showing proliferation of HER14 cells treated withcyclic peptides of the invention;

FIG. 14a is a bar graph showing proliferation of SupM2 cells treatedwith peptides of the invention;

FIG. 14b is a bar graph showing proliferation of SupM2 cells treatedwith peptides of the invention;

FIG. 15a is an immunoblot showing MAPK activation on PC12 cells treatedwith peptides of the invention;

FIG. 15b is an immunoblot showing MAPK activation on PC12 cells treatedwith peptides of the invention;

FIG. 16a is an immunoblot showing activated MAPK on PC12 cells treatedwith peptides of the invention; and

FIG. 16b is an immunoblot showing activated MAPK on PC12 cells treatedwith peptides of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The following standard abbreviations for the amino acid residues areused throughout the specification: A, Ala-alanine; C, Cys-cysteine; D,Asp-aspartic acid; E, Glu-glutamic acid; F, Phe-phenylalanine; G,Gly-glycine; H, His-histidine; I, Ile-isoleucine; K, Lys-lysine; L,Leu-leucine; M, Met-methionine; N, Asn-asparagine; P, Pro-proline; Q,Gln-glutamine; R, Arg-arginine; S, Ser-serine; T, Thr-threonine; V,Val-valine; W, Trp-tryptophan; Y, Tyr-tyrosine; and p.Y.,P.Tyr-phosphotyrosine.

As mentioned previously, the present invention relates to a peptide ofthe formula I

    X.sup.1 -A.sup.1 -A.sup.2 -X.sup.2 -Asn-X.sup.3 -X.sup.4 -P.Tyr-X.sup.5 -X.sup.6 -X.sup.7 -X.sup.8                                I

wherein X¹ represents Lys, Arg, His, Ser, Thr, Tyr, Asn, Leu, Val, orGlu, A¹ represents Trp, Leu, Ala, Ser, Ile, Glu, Met, Gly, Cys, Phe,Pro, or Val, and A² represents Ala, Val, Leu, Ile, Ser, Met, Phe, Gly,Cys, Trp, or Pro, X² represents Glu, Asn, Tyr, Thr, Ser, Asp, or Ile, X³represents Pro, Met, Trp, Phe, Ala, Lys, Val, Leu, Ile, Gly, or Cys, X⁴represents Leu, Ala, Glu, Gin, Asp, Asn, Tyr, Thr, or Ser, X⁵ representsPhe, Trp, Pro, Leu, Ala, Val, Ile, Gly, Cys, Met, Arg or Ser, X⁶represents Ser, Thr, Tyr, Asn, Glu, Met, Ala, Leu, Val, or Gly, X⁷represents Asp, Gin, Ala, Val, Leu, Ile, Gly, Cys, Phe, Trp, Met, Pro,Ser, or Asn, and X⁸ which may be present or absent represents Phe, Trp,Pro, Leu, Ala, Val, Ile, Gly, Cys, Met, Asp, Ser, or Arg, whichinterferes with the interaction of a PTB domain containing protein witha PTB domain binding site.

In an embodiment of the present invention a peptide of the formula I isprovided:

    X.sup.1 -A.sup.1 -A.sup.2 -X.sup.2 -Asn-X.sup.3 -X.sup.4 -P.Tyr -X.sup.5 -X.sup.6 -X.sup.7 -X.sup.8                                I

wherein X¹ represents His, Ser, Thr, Tyr, Asn, Leu, Val, or Gin, X²represents Glu, Ser, Asp, or Ile, X³ represents Pro or Lys, X⁴represents Leu, Ala, Glu, Gln Asn, or Thr, X⁵ represents Phe, Leu, Ile,Gly, Arg, or Ser, X⁶ represents Ser, Thr, Met, Ala, Leu, Val, or Gly, X⁷represents Asp, Ala, Val, Leu, Met, Ser, or Asn, X⁸ which may be presentor absent, represents Leu, Ala, Gly, Asp, Ser, or Arg, A¹ representsTrp, Leu, Ala, Ser, Ile, Gln, Met, or Val, and A² represents Ala, Val,Leu, Ile, Ser, Met, or Phe, which interferes with the interaction of aPTB domain containing protein with a PTB domain binding site.

In another embodiment of the invention a peptide of the formula Ia isprovided

    X.sup.1 -A.sup.1 -A.sup.2 -X.sup.2 -Asn-X.sup.3 -X.sup.4 -P.Tyr-X.sup.5 -X.sup.6 -X.sup.7                                         Ia

wherein X¹ represents Lys, Arg, His, preferably His, X² represents Glu,Asn, Tyr, Thr, Ser, preferably Glu, X³ represents Pro, Met, Trp, Phe,Ala, Val, Leu, Ile, Gly, Cys, preferably Pro, X⁴ represents Gln, Asp,Asn, Tyr, Thr, Ser, preferably Gln, X⁵ represents Phe, Trp, Pro, Leu,Ala, Val, Ile, Gly, Cys, Met, preferably Phe, X⁶ represents Ser, Thr,Tyr, Asn, Gln, preferably Ser, X⁷ represents Asp, Gin, preferably Asp,and one of A¹ and A² represents Ile and the other of A¹ and A²represents Ile or Ala, preferably A¹ represents Ala and A² representsIle, which interferes with the interaction of a PTB domain containingprotein with a PTB domain binding site.

In still another embodiment of the invention a peptide of the formula Iais provided

    X.sup.1 -A.sup.1 -A.sup.2 -X.sup.2 -Asn-X.sup.3 -X.sup.4 -P.Tyr-X.sup.5 -X.sup.6 -X.sup.7                                         Ia

wherein X¹ represents Ser, Thr, Tyr, Asn or Glu, preferably Tyr, X²represents Glu, Asn, Tyr, Thr, Ser, preferably Ser, X³ represents Pro,Met, Trp, Phe, Ala, Val, Leu, Ile, Gly, Cys, preferably Pro, X⁴represents Glu, Asp, preferably Glu, X⁵ represents Phe, Trp, Pro, Leu,Ala, Val, Ile, Gly, Cys, Met preferably Leu, X⁶ represents Ser, Thr,Tyr, Asn, Glu, preferably Ser, X⁷ represents Ala, Val, Leu, Ile, Gly,Cys, Phe, Trp, Met, Pro, preferably Ala, and one of A¹ and A² representsIle and the other of A¹ and A² represents Ala, Val, Leu, Ile, Gly, Cys,Phe, Trp, Met or Pro, which interferes with the interaction of a PTBdomain containing protein with a PTB domain binding site.

Preferred peptides of the invention include the following:His-Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp;His-Ala-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp;His-Ile-Ala-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp;Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp-Ala;Tyr-Ala-Ile-Ser-Asn-Pro-Glu-P.Tyr-Leu-Ser-Ala;Thr-Trp-Ile-Glu-Asn-Lys-Leu-P.Tyr-Gly-Met-Ser-Asp;Thr-Trp-Ile-Glu-Asn-Lys-Leu-P.Tyr-Gly-Thr-Ser-Asp;Leu-Leu-Leu-Ser-Asn-Pro-Ala-P.Tyr.-Arg-Leu-Leu-Leu;Tyr-Ala-Ser-Ser-Asn-Pro-Glu-P.Tyr-Leu-Ser-Ala-Ser;Val-Ser-Val-Asp-Asn-Pro-Glu-P.Tyr-Leu-Leu-Asn-Ala;Ser-Leu-Leu-Ser-Asn-Pro-Thr-P.Tyr-Ser-Val-Met-Arg;Asn-Glu-Met-Ile-Asn-Pro-Asn-P.Tyr-Ile-Gly-Met-Gly; andGlu-Met-Phe-Glu-Asn-Pro-Leu-P.Tyr-Gly-Ser-Val-Ser (SEQ. ID. NOS. 1-13 inthe Sequence Listing).

In addition to full-length peptides of the formula I, truncations of thepeptides which inhibit interaction of PTB domain containing proteinswith PTB domain binding sites are contemplated in the present invention.Truncated peptides may comprise peptides of about 7 to 10 amino acidresidues. In an embodiment of the invention the truncated peptide hasthe sequence A² -X² -Asn-X³ -X⁴ -P.Tyr or A² -X² -Asn-X³ -X⁴ -P.Tyr-X⁵wherein A², X², X³, X⁴, and X⁵ are as defined above. In a preferredembodiment of the invention, the truncated peptide has the sequenceLeu/Ile-X² -Asn-Pro-X⁴ -P.Tyr, wherein X² represents Glu, Ser, Asp, orIle, and X⁴ represents Leu, Ala, Glu, Gln, Asn, or Thr. Examples oftruncated peptides include Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe;Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Pro-Gly;Ala-Glu-Asn-Pro-Gln-P.Tyr-Phe; Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser;Ile-Ser-Asn-Pro-Glu-P.Tyr-Leu;Val-Leu-Ala-Asp-Asn-Pro-Ala-P.Tyr-Arg-Ser-Ala (SEQ. ID. NOs. 14 to 19 inthe Sequence Listing).

The truncated peptides may have an amino group (--NH₂), a hydrophobicgroup (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), anacetyl group, a 9-fluorenylmethoxy-carbonyl (PMOC) group, or amacromolecule including but not limited to lipid-fatty acid conjugates,polyethylene glycol, or carbohydrates at the amino terminal end. Thetruncated peptides may have a carboxyl group, an amido group, aT-butyloxycarbonyl group, or a macromolecule including but not limitedto lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates atthe carboxy terminal end.

The peptides of the invention may also include analogs of the peptide ofthe Formula I, and/or truncations of the peptide, which may include, butare not limited to the peptide of the formula I containing one or moreamino acid insertions, additions, or deletions, or both. Analogs of thepeptide of the invention exhibit the activity characteristic of thepeptide i.e. interference with the interaction of a PTB domaincontaining protein with a PTB domain binding site, and may furtherpossess additional advantageous features such as increasedbioavailability, stability, or reduced host immune recognition.

One or more amino acid insertions may be introduced into a peptide ofthe formula I preferably outside the sequence A² -X² -Asn-X³ -X⁴-P.Tyr-X⁵. For example, amino acid insertions may be made between X¹ andA¹ or between X⁵ and X⁶, or X⁶ and X⁷. Amino acid insertions may consistof a single amino acid residue or sequential amino acids.

One or more amino acids, preferably one to five amino acids, may beadded to the right or left termini of a peptide of the invention.Examples of such analogs includeAla-Leu-Leu-Leu-Ser-Asn-Pro-Ala-P.Tyr.-Arg-Leu-Leu-Leu-Ala;Gly-Pro-Leu-Tyr-Ala-Ser-Ser-Asn-Pro-Glu-P.Tyr-Leu-Ser-Ala-Ser-Asp-Val-Phe;Pro-Val-Ser-Val-Asp-Asn-Pro-Glu-P.Tyr-Leu-Leu-Asn-Ala-Gln-Lys;Leu-Ser-Leu-Leu-Ser-Asn-Pro-Thr-P.Tyr-Ser-Val-Met-Arg-Ser-Lys;Val-Ser-Ser-Leu-Asn-Glu-Met-Ile-Asn-Pro-Asn-P.Tyr-Ile-Gly-Met-Gly-Pro-Phe;andLeu-Leu-Leu-Thr-Lys-Pro-Glu-Met-Phe-Glu-Asn-Pro-Leu-P.Tyr-Gly-Ser-Val-Ser-Ser-Phe(SEQ. ID. NOs. 20 to 25 in the Sequence Listing).

Deletions may consist of the removal of one or more amino acids, ordiscrete portions from the peptide sequence preferably outside the A²-X² -Asn-X³ -X⁴ -P.Tyr sequence. The deleted amino acids may or may notbe contiguous. The lower limit length of the resulting analog with adeletion mutation is about 7 amino acids.

It is anticipated that if amino acids are inserted or deleted insequences outside the A² -X² -Asn-X³ -X⁴ -P.Tyr sequence that theresulting analog of the peptide will exhibit the activity of a peptideof the invention.

Cyclic derivatives of the peptides of the invention are also part of thepresent invention. Cyclization may allow the peptide to assume a morefavorable conformation for association with a PTB domain containingprotein. Cyclization may be achieved using techniques known in the art.For example, disulfide bonds may be formed between two appropriatelyspaced components having free sulfhydryl groups, or an amide bond may beformed between an amino group of one component and a carboxyl group ofanother component. Cyclization may also be achieved using anazobenzene-containing amino acid as described by Ulysse, L., et al., J.Am. Chem. Soc. 1995, 117, 8466-8467. The side chains of P.Tyr and Asnmay be linked to form cyclic peptides. The components that form thebonds may be side chains of amino acids, non-amino acid components or acombination of the two.

Preferred cyclic peptides of the invention includecyclo-(Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Pro-Gly, andcyclo-(Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp-Ala-Pro-Gly) (SEQ. ID.NOs. 26 to 27 in the Sequence Listing) where the amino group ofisoleucine and the carboxyl group of glycine form a peptide bond;cyclo-(His-Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp-Ala-Pro-Gly) (SEQ.ID. NO. 28 in the Sequence Listing) where the amino group of histidineand the carboxyl group of glycine form a peptide bond; andCys-Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Cys (SEQ. ID. NO. 29 in theSequence Listing) having a disulphide bond between the two cysteineresidues.

In an embodiment of the invention, cyclic peptides are contemplated thathave a beta-turn in the right position. Beta-turns may be introducedinto the peptides of the invention by adding the amino acids Pro-Gly atthe right position. An example of such a cyclic peptide is a peptide ofthe invention with an Ile in the left position (i.e. a terminal A¹ or A²is Ile) and the amino acids Pro-Gly at the right position. The aminogroup of the Ile and the carboxyl group of the Gly form a peptide bondresulting in a cyclic peptide. The 3D structure of the cyclic peptide issimilar to the original structure of the PTB binding site of TrkA.

The following is an example of a cyclic peptide that has a beta-turn inthe right position: cyclo-(Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Pro-Gly)(SEQ. ID. NO. 26 in the Sequence Listing). In this peptide,Asn-Pro-Gln-P.Tyr [aa 3-6 of SEQ ID NO:26] take a native beta-turn,Ser-Pro-Gly-Ile [aa 8, 9, 10, and 1 of the cyclopeptide of SEQ ID NO:26]make another beta-turn on the other side, and the central part adopts anantiparallel beta-sheet. A beta-sheet has two faces, and the peptidebinds to the PTB domain with the face on which the side chains of Ile,Asn, and P.Tyr extend. The side chains of Glu and Phe are on the otherface, and may not affect the binding affinity. It may be possible tocontrol the binding specificity by the side-chain of Gln as this sidechain may contact the PTB domain.

It may be desirable to produce a cyclic peptide which is more flexiblethan the cyclic peptides containing peptide bond linkages as describedabove. A more flexible peptide may be prepared by introducing cysteinesat the right and left position of the peptide and forming a disulphidebridge between the two cysteines. The two cysteines are arranged so asnot to deform the beta-sheet and turn. The peptide is more flexible as aresult of the length of the disulfide linkage and the smaller number ofhydrogen bonds in the beta-sheet portion. The relative flexibility of acyclic peptide can be determined by molecular dynamics simulations.

The invention also includes a peptide conjugated with a selectedpeptide, protein, or a selectable marker (see below) to produce fusionproteins. For example, a peptide of the invention may be conjugated witha peptide which facilitates entry into cells.

The peptides of the invention may be converted into pharmaceutical saltsby reacting with inorganic acids such as hydrochloric acid, sulfuricacid, hydrobromic acid, phosphoric acid, etc., or organic acids such asformic acid, acetic acid, propionic acid, glycolic acid, lactic acid,pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid,citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, andtoluenesulfonic acids.

The peptides of the invention may be prepared using recombinant DNAmethods. Accordingly, nucleic acid molecules which encode a peptide ofthe invention may be incorporated in a known manner into an appropriateexpression vector which ensures good expression of the peptide. Possibleexpression vectors include but are not limited to cosmids, plasmids, ormodified viruses so long as the vector is compatible with the host cellused. The expression vectors contain a nucleic acid molecule encoding apeptide of the invention and the necessary regulatory sequences for thetranscription and translation of the inserted protein-sequence. Suitableregulatory sequences may be obtained from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes (Forexample, see the regulatory sequences described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Selection of appropriate regulatory sequences isdependent on the host cell chosen, and may be readily accomplished byone of ordinary skill in the art. Other sequences, such as an origin ofreplication, additional DNA restriction sites, enhancers, and sequencesconferring inducibility of transcription may also be incorporated intothe expression vector.

The recombinant expression vectors may also contain a selectable markergene which facilitates the selection of transformed or transfected hostcells. Suitable selectable marker genes are genes encoding proteins suchas G418 and hygromycin which confer resistance to certain drugs,β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase,or an immunoglobulin or portion thereof such as the Fc portion of animmunoglobulin preferably IgG. The selectable markers may be introducedon a separate vector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes which encode afusion portion which provides increased expression of the recombinantpeptide; increased solubility of the recombinant peptide; and/or aid inthe purification of the recombinant peptide by acting as a ligand inaffinity purification. For example, a proteolytic cleavage site may beinserted in the recombinant peptide to allow separation of therecombinant peptide from the fusion portion after purification of thefusion protein. Examples of fusion expression vectors include pGEX(Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the recombinant protein.

Recombinant expression vectors may be introduced into host cells toproduce a transformant host cell. Transformant host cells includeprokaryotic and eukaryotic cells which have been transformed ortransfected with a recombinant expression vector of the invention. Theterms "transformed with", "transfected with", "transformation" and"transfection" are intended to include the introduction of nucleic acid(e.g. a vector) into a cell by one of many techniques known in the art.For example, prokaryotic cells can be transformed with nucleic acid byelectroporation or calcium-chloride mediated transformation. Nucleicacid can be introduced into mammalian cells using conventionaltechniques such as calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofectin,electroporation or microinjection. Suitable methods for transforming andtransfecting host cells may be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratorypress (1989)), and other laboratory textbooks.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the peptides of the invention may be expressedin bacterial cells such as E. coli, insect cells (using baculovirus),yeast cells or mammalian cells. Other suitable host cells can be foundin Goeddel, Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1991).

The peptides of the invention may be tyrosine phosphorylated using themethod described in Reedijk et al. (The EMBO Journal 11(4): 1365, 1992).For example, tyrosine phosphorylation may be induced by infectingbacteria harbouring a plasmid containing a nucleotide sequence encodinga peptide of the invention, with a λgt11 bacteriophage encoding thecytoplasmic domain of the Elk tyrosine kinase as a LacZ-Elk fusion.Bacteria containing the plasmid and bacteriophage as a lysogen areisolated. Following induction of the lysogen, the expressed peptidebecomes phosphorylated by the Elk tyrosine kinase.

The peptides of the invention may also be prepared by chemical synthesisusing techniques well known in the chemistry of proteins such as solidphase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85: 2149-2154) orsynthesis in homogenous solution (Houbenweyl, 1987, Methods of OrganicChemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart). By wayof example, the peptides may be synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid phase chemistry with direct incorporationof phosphotyrosine as the N-fluorenylmethoxy-carbonyl-O-dimethylphosphono-L-tyrosine derivative.

N-terminal or C-terminal fusion proteins comprising a peptide of theinvention conjugated with other molecules may be prepared by fusing,through recombinant techniques, the N-terminal or C-terminal of thepeptide, and the sequence of a selected protein or selectable markerwith a desired biological function. The resultant fusion proteinscontain the peptide fused to the selected protein or marker protein asdescribed herein. Examples of proteins which may be used to preparefusion proteins include immunoglobulins, glutathione-S-transferase(GST), hemagglutinin (HA), and truncated myc.

The peptides of the invention may be used to prepare monoclonal orpolyclonal antibodies. Conventional methods can be used to prepare theantibodies. As to the details relating to the preparation of monoclonalantibodies reference can be made to Goding, J. W., MonoclonalAntibodies: Principles and Practice, 2nd Ed., Academic Press, London,1986. As discussed below, the antibodies may be used to identifyproteins with PTB domain binding sites.

The peptides and antibodies specific for the peptides of the inventionmay be labelled using conventional methods with various enzymes,fluorescent materials, luminescent materials and radioactive materials.Suitable enzymes, fluorescent materials, luminescent materials, andradioactive material are well known to the skilled artisan. Labeledantibodies specific for the peptides of the invention may be used toscreen for proteins with PTB domain binding sites, and labeled peptidesof the invention may be used to screen for PTB domain containingproteins such as Shc.

The peptides of the invention interfere with the interaction of a PTBdomain containing protein and a PTB domain binding site. The term "PTBdomain containing protein" refers to a protein or peptide whichcomprises or consists of a PTB domain. A PTB domain is a region which isa domain of ˜160 amino acids which was originally identified in Shc andSck (Kavanaugh, V. M. Et al., 1995 Science, 268: 1177-1179; Bork, R P,and Margolis, B, Cell, Vol 80: 693-694, 1995; Craparo, A., et al., 1995,J. Biol. Chem. 270: 15639-15643; van der Geer, P., & Pawson, T., 1995,TIBS 20: 277-280; Batzer, A. G., et al., Mol. Cell. Biol. 1995, 15:4403-4409; and Trub, T., et al., 1995, J. Biol. Chem. 270: 18205-18208;van der Geer et al., Current Biology 5(4): 404, 1995)). The PTB domaincomprises residues 46 to 208 in the 52 kDa isoform of Shc. The sequencesof several known PTB domains are aligned in FIG. 7. In FIG. 7, residuesthat are conserved within the sequences are shaded.

Examples of PTB domain containing proteins are mammalian Shc and Sck,IRS-1, and homologues of Shc including Drosophila Shc, and mouse Shc.Other proteins that contain homologous PTB domains have been identifiedusing data base search methods (Bork, R. P., and Margolis, B. Cell, Vol80: 693-694, 1995). PTB domain containing proteins may also beidentified by screening a cDNA expression library with a proteincontaining a sequence with high affinity to PTB domains, i.e. a PTBdomain binding sequence or a peptide of the invention which may belabeled. PTB domain containing proteins may also be screened usingantibodies specific for the PTB domain. For example, a PTB domain thatbinds to the consensus sequence Leu/Ile-X-Asn-Pro-X-P.Tyr found ingrowth factors may be identified by screening a cDNA expression librarywith proteins based on the consensus sequence. PCR(Wilks, A. F., Proc.Natl. Acad. Sci. U.S.A. Vol. 86, pp. 1603-1607, March 1989) or lowstringency screening (Hanks, S. K., Proc. Natl. Acad. Sci. U.S.A. Vol.84, pp 388-392, January 1987) with the PTB domain specific probe can beused.

The term "PTB domain binding site" refers to a sequence with highaffinity to PTB domains. PTB domain binding sequences have beenidentified in activated growth factors such as activated nerve growthfactor receptor, activated epidermal growth factor (EGF) receptor,polyoma middle T antigen, and SHIP (Blaikie et al., 1994; Kavanaugh andWilliams, 1994; van der Geer et al., 1995; Damen et al., 1996), ErbB2,ErbB3, TrkA, TrkB, TrkC, MCK10b, insulin receptor, IGF-1 receptor, andIL-4 receptor. PTB domain binding sites may be identified by screeningwith PTB domain containing proteins or with antibodies specific for thepeptides of the invention.

The phrase "interfere with the interaction of" refers to the ability ofthe peptides of the invention to inhibit the binding of a PTB domaincontaining protein to a PTB domain binding site thereby affectingregulatory pathways that control gene expression, cell division,cytoskeletal architecture and cell metabolism. Examples of suchregulatory pathways are the Ras pathway, the pathway that regulates thebreakdown of polyphosphoinositides through phospholipase C, andPI-3-kinase activated pathways, such as the rapamycin-sensitive proteinkinase B (PKB/Akt) pathway.

The peptides of the invention have been specifically shown to interferewith the interaction of the PTB domain of Shc andphosphotyrosine-containing peptides based on the sequence around Tyr 490in activated nerve growth factor receptor and based on the Shc bindingsite in polyoma middle T antigen. Accordingly, the activity of a peptideof the invention may be confirmed by assaying for the ability of thepeptide to interfere with the interaction of the PTB domain of Shc andphosphotyrosine-containing peptides based on the sequence around Tyr 490in activated nerve growth factor receptor, or based on the Shc bindingsite in polyoma middle T antigen.

Computer modelling techniques known in the art may also be used toobserve the interaction of a peptide of the invention, and truncationsand analogs thereof with a PTB domain containing protein (for example,Homology Insight II and Discovery available from BioSym/MolecularSimulations, San Diego, Calif., U.S.A.). If computer modelling indicatesa strong interaction, the peptide can be synthesized and tested for itsability to interfere with the binding of the PTB domain of Shc andphosphotyrosine-containing peptides as discussed above.

The peptides of the invention mediate the interactions of PTB domaincontaining proteins with PTB domain binding sites on proteins such asgrowth factors and cytokine receptors which regulate pathways thatcontrol gene expression, cell division, cytoskeletal architecture andcell metabolism. The peptides may therefore be used in the treatment ofconditions involving perturbation of such regulatory pathways. Inparticular, the peptides may be useful in treating disorders involvingexcessive proliferation including various forms of cancer such asleukemias, lymphomas (Hodgkins and non-Hodgkins), sarcomas, melanomas,adenomas, carcinomas of solid tissue, hypoxic tumors, squamous cellcarcinomas of the mouth, throat, larynx, and lung, genitourinary cancerssuch as cervical and bladder cancer, hematopoietic cancers, head andneck cancers, and nervous system cancers, ovarian cancer, breast cancer,glioblastoma, benign lesions such as papillomas, arthrosclerosis,angiogenesis, and viral infections, in particular HIV infections; andautoimmune diseases including systemic lupus erythematosus, Wegener'sgranulomatosis, rheumatoid arthritis, sarcoidosis, polyarthritis,pemphigus, pemphigoid, erythema multiforme, Sjogren's syndrome,inflammatory bowel disease, multiple sclerosis, myasthenia gravis,keratitis, scleritis, Type I diabetes, insulin-dependent diabetesmellitus, Lupus Nephritis, and allergic encephalomyelitis.

The invention also relates to a pharmaceutical composition comprising apeptide of the invention for use as an antagonist of the interaction ofa PTB domain containing protein, preferably Shc and a PTB domain bindingsite, preferably an activated growth factor or cytokine receptor.

The peptides of the invention may be formulated into pharmaceuticalcompositions for adminstration to subjects in a therapeutically activeamount and in a biologically compatible form suitable for administrationin vivo i.e. a form of the peptides to be administered in which anytoxic effects are outweighed by the therapeutic effects.

The peptides may be administered to living organisms including humans,and animals. A therapeutically active amount of the pharmaceuticalcompositions of the invention is defined as an amount effective, atdosages and for periods of time necessary to achieve the desired result.For example, a therapeutically active amount of a peptide may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual. Dosage regime may be adjusted to provide the optimumtherapeutic response.

The peptides may be administered in a convenient manner such as byinjection (subcutaneous, intravenous, etc.), oral administration,inhalation, transdermal application, or rectal administration. Dependingon the route of administration, the peptides may be coated in a materialto protect them from the action of enzymes. The peptides may also beused in combination with organic substances for prolongation of theirpharmacologic actions. Examples of such organic substances arenon-antigenic gelatin, carboxymethylcellulose, sulfonate or phosphateester of alginic acid, dextran, polyethylene glycol and other glycols,phytic acid, polyglutarnic acid, and protamine.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to subjects, such that an effective quantityof a peptide is combined in a mixture with a pharmaceutically acceptablevehicle. Suitable vehicles are described, for example, in Remington'sPharmaceutical Sciences (Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa., USA 1985). On this basis, thecompositions include, albeit not exclusively, solutions of the peptidesin association with one or more pharmaceutically acceptable vehicles ordiluents, and contained in buffered solutions with a suitable pH andiso-osmotic with the physiological fluids. The peptides may also beincorporated in liposomes or similar delivery vehicles.

The utility of the peptides and compositions of the invention may beconfirmed in in vitro cell penetration assays. For example, the effectsof the peptides upon cellular functions in vivo may be confirmed usingelectroporation techniques (See Raptis, L., and K. L. Firth, DNA andCell Biology, 9: 615, 1990 and Raptis, L. H. Et al., BioTechniques 18:104, 1995).

The utility of the peptides and compositions of the invention may alsobe confirmed in in vivo animal experimental model systems. For example,therapeutic utility in proliferative disorders may be tested byexamining the ability of a substance to suppress the growth of atransplantable tumor. Particular in vivo animal models which may be usedinclude the growth of human tumor cell lines (e.g. glioblastomas) innude mice; and the development of tumors in mice that carryMMTV-polyomavirus middle T antigen or MMTV-neu transgenes, which resultin the development of mammary carcinoma.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1

The following materials and methods were utilized in the investigationsoutlined in the example:

Materials and Methods

Cell lines, anti-sera and fusion proteins

CHO cells expressing Wt insulin receptors (White et al., 1988) weregrown in F12 medium containing 25 mM Hepes pH 7.4, and 10% fetal bovineserum. NIH3T3 cells expressing Wt and Phe 490 mutant NGF receptor(Stephens et al., 1994) were grown Dulbecco-Vogt's modified Eagle medium(DMEM) containing 10% calf serum (CS). NIH3T3 cells overexpressing thehuman EGF receptor (Honegger et al., 1987) were grown in DMEM containing10% CS and 400 μg/ml G418. The monoclonal anti-insulin receptor antibody51 was obtained from Dr. I. Goldfine (Forsayeth et al., 1987; Roth etal., 1982). A polyclonal anti-NGF receptor antiserum was raised againstNGF receptor carboxy-terminus (Hempstead et al., 1992), the anti-Shcpolyclonal serum was raised against a GST-Shc SH2 domain fusion protein.The anti-P.Tyr monoclonal Antibody 4G10 was obtained from UBI (LakePlacid N.Y.). The GST-Shc PTB fusion protein used in the receptorbinding experiments described here is identical to GST-ShcB described invan der Geer et al., 1995. The GST-dShc PTB fusion protein has beendescribed previously (Lai et al., 1995).

Immunoprecipitations and PTB Binding Assays

Cells were grown to confluence and starved 16 hr in medium withoutserum. CHO cells expressing the insulin receptor were stimulated with100 nM insulin for 5 min at 37° C. NIH3T3 cells expressing NGF receptorswere stimulated with 50 ng/ml NGF for 5 min at 37° C., and NIH3T3 cellsexpressing the human EGF receptor were stimulated with 100 ng/ml EGF for5 min at 37° C. Control and growth factor stimulated cells were rinsedtwice with cold PBS and lysed in 1 ml 50 mM Hepes pH 7.5, 150 mM NaCl,10% glycerol, 1% Triton X100, 1.5 mM MgCl₂, 1 mM EGTA, 100 mM NaF, 10 mMSodium Pyrophosphate, 500 μM Sodium Vanadate, 1 mM PMSF, 10 μg/mlAprotinin, and 10 μg/ml Leupeptin (PLC-lysis buffer) per 10 cm dish.Immunoprecipitations and PTB-binding assays in the absence or presenceof 2 or 5 μM competing phosphopeptide were performed exactly asdescribed previously (van der Geer et al., 1995).

Surface Plasmon Resonance Analysis of Phosphopeptides Interacting withthe Shc PTB Domain

Peptides were synthesized using 9-fluorenyl methoxycarbonyl (Fmoc) solidphase chemistry with direct incorporation of phosphotyrosine as theN-fluorenylmethoxy-carbonyl-O-dimethyl-phosphono-L-tyrosine derivative.Cleavage of the peptide from the resin and deprotection was achievedthrough an 8 hr incubation at 4° C. in trifluoroacetic acid containing 2M bromotrimethyl silane and a scavenger mixture composed of thioanisole,m-cresol and 1,2-ethanedithiol (1.0:0.5:0.1% by volume). The product wasprecipitated with cold t-butyl ethylether and collected bycentrifugation. Following desalting of the crude material, purephosphopeptide was isolated using reverse phase HPLC. The authenticityof the phosphopeptide was confirmed by amino acid analysis and massspectroscopy.

Surface plasmon resonance analysis was carried out using a Biacoreapparatus (Pharmacia Biosensor) as described previously (Puil et al.,1994). The peptide L-S-L-L-S-N-P-T-p.Y-S-V-M-R-S-K (SEQ ID NO:23) wasimmobilized to a biosensor chip through injection of a 0.5 mM solutionof the phosphopeptide, in 50 mM HEPES, pH 7.5 and 2 M NaCl, across thechip surface previously activated following procedures outlined by themanufacturer. Injection of anti-phosphotyrosine antibody was used toconfirm that successful immobilization of the peptide was achieved.Solutions (100 μl) containing 1 μM GST-Shc PTB domain fusion protein andthe indicated concentrations of soluble phosphopeptide in 50 mM Naphosphate, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, and 2 mM DTT, were injectedacross the surface. The amount of bound GST-Shc PTB domain was estimatedfrom the surface plasmon resonance signal at a fixed time following theend of the injection and the percentage bound, relative to injection ofGST-Shc PTB domain alone, calculated. The surface was regenerated using2 M Guanidinium-HCl.

Expression of Torso-DER in Transgenic Flies

Transgenic flies expressing the activated Torso-DER chimeric proteinexpressed under the control of the heat shock promoter were obtained andprotein expression was induced by growing the flies at 37° C. for 45 minafter which they were allowed to recover at room temperature for 2.5 hr.Lysates were made as described before (Lai et al., 1995).

I. Identifying Motifs Recognized by the Shc PTB Domain

The PTB domain was found to bind tyrosine phosphorylated proteins thatcontain phosphorylation sites present within the sequenceAsn-Pro-X-P.Tyr. To confirm that it is indeed the Asn-Pro-X-P.Tyr motifthat is recognized by the PTB domain, it was shown that peptides thatcontain a phosphotyrosine within the sequence Asn-Pro-X-P.Tyr cancompete for binding of the Shc PTB domain to activated growth factorreceptors. The specificity was confirmed by sequencing peptides presentin a degenerate phosphopeptide library that bind to the Shc PTB domain(Songyang et al., 1995). To investigate the contribution of the Asn andPro residues within the consensus PTB domain binding site tophosphopeptide recognition, the residues were changed to Ala in aphosphopeptide based on the sequence around Tyr 490, the Shc-bindingsite in the NGF receptor. Wt and mutant peptides were tested for theirability to compete with NGF receptors, present in lysates ofNGF-stimulated cells, for binding to a GST fusion protein containing theShc PTB domain (FIG. 1A). Bound proteins were detected by anti-P.Tyrimmunoblotting. Only the activated NGF receptor bound the Shc PTB domainin vitro. The Wt phosphopeptide(His-Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp, SEQ ID NO:1) competedefficiently for binding. Changing the Asn at position -3 (relative tothe P.Tyr) to Ala completely abolished binding, whereas changing the Proat -2 to Ala reduced the affinity of the PTB-peptide interaction. Theidentity of the NGF receptor was confirmed by stripping and reprobingthe blot with a polyclonal antiserum raised against the NGF receptor(FIG. 1B). To confirm these results and to estimate the contribution ofthe different residues more precisely, a wide range of concentrations ofthe different peptides was tested for their ability to inhibit bindingof the Shc PTB domain to a phosphotyrosine-containing peptide, based onthe sequence around Tyr 250 the Shc-binding site in polyoma middle Tantigen, immobilized on a Biacore chip (FIG. 2). The data show thatwithin the Asn-Pro-X-P.Tyr motif the Asn residue is essential forpeptide binding by the PTB domain; presence of the Pro residue furtherincreases the affinity approximately ten fold (Table 1). PTB bindingdepends on phosphorylation of the Tyr residue present within theconsensus binding site (Blaikie et al., 1994; Kavanaugh and Williams,1994; van der Geer et al., 1995). These results are consistent with thepresence of Asn-Pro-X-P.Tyr motifs in a variety of receptors for growthfactors and cytokines that have been shown to bind Shc.

II. Why the Insulin Receptor Lacks the Ability to bind to Shc

The insulin receptor, which contains a bona fide autophosphorylationsite that is present within the sequence Asn-Pro-Glu-P.Tyr (SEQ IDNO:32), lacks the ability to bind to Shc (Kovacina and Roth, 1993; Pronket al., 1993). Tyr 960 in the insulin receptor is present in the juxtamembrane domain, between the membrane and the kinase domain, in aposition very similar to Tyr 490 in the NGF receptor (see FIG. 3). Theinability of the insulin receptor to associate stably with Shc wasconfirmed in coimmunoprecipitation experiments in which Shcimmunoprecipitates were analyzed for associated proteins by anti-P.Tyrimmunoblotting (FIG. 4A). Wt but not Phe 490 mutant NGF receptors can bedetected in Shc immunoprecipitates from NGF-stimulated cells. Incontrast, insulin receptors were absent from Shc immunoprecipitates frominsulin stimulated CHO cells overexpressing the Wt insulin receptor(CHO-IR cells). To test whether the insulin receptors inability toassociate with Shc in cells was reflected in an inability to bind to theShc PTB domain in vitro, GST fusion proteins containing the Shc PTBdomain were incubated with lysates of control and insulin-stimulatedCHO-IR cells and bound proteins were visualized by anti-P.Tyrimmunoblotting. Wt and Phe 490 NGF receptors were included as controls.The NGF receptor bound to the Shc PTB domain in vitro (FIG. 4B) andbinding was dependent on phosphorylation of the NGF receptor at Tyr 490.In contrast, no tyrosine phosphorylated insulin receptors were bound tothe Shc PTB domain in vitro (FIG. 4B).

To investigate the possibility that access to the Asn-Pro-X-P.Tyr motifin the insulin receptor is blocked, the ability of thephosphotyrosine-containing peptide based on the sequence around Tyr 960in the insulin receptor to compete with the NGF receptor for binding tothe Shc PTB domain was tested. In contrast to the NGF receptorphosphopeptide, the insulin receptor peptide was unable to compete (FIG.5A, lanes 2 and 6, and FIG. 5B). This indicates that the inability ofthe insulin receptor to bind the PTB domain is retained in thisphosphopeptide that starts seven amino acid residues amino-terminal tothe P.Tyr (Table 1). The NGF receptor and several other proteins withwell defined Shc-binding sites often contain large aliphatic residues atsix and five residues amino-terminal of the phosphorylated Tyr residue.These large aliphatic residues are absent from the insulin receptor,which has an Ala and a Ser six and five residues amino-terminal to Tyr960 (Table 1). To test the possibility that these residues are importantfor PTB binding, several substitutions at these positions were made inthe NGF receptor peptide and mutant peptides were tested for theirability to block binding of the PTB domain to the NGF receptor and tothe polyoma middle T antigen phosphopeptide. Changing the Ile sixresidues upstream of the P.Tyr to an Ala had no effect on the ability tobind to the PTB domain (FIGS. 5A and 5B). In contrast, changing the Ileat -5 to Ala in addition to changing the Ile at -6 reduced the abilityto bind to the PTB domain (FIGS. 5A and 5B). Changing the Ile residuesat -5 to a Ser in addition to changing the Ile at -6 to Ala, identicalto what is found in the insulin receptor, abolished binding (FIGS. 5Aand 5B). These data clearly implicate the aliphatic residues five andsix residues amino-terminal to the phosphotyrosine in the PTB-bindingsite as being important for binding to the Shc PTB domain and suggestthat changing the Ser, five residues upstream of the P.Tyr in theinsulin receptor peptide, to an Ile should increase its ability to bindto the PTB domain dramatically. This was tested and it was found that incontrast to the Wt insulin receptor peptide, which has no measurableaffinity for the PTB domain, the mutant insulin receptor peptidecompeted efficiently with the NGF receptor and the NGF receptor peptidefor binding to the PTB domain (FIGS. 5A and 5B). The data presented here(summarized in Table 1) indicate that the Shc PTB domain specificallyrecognizes P.Tyr residues in the context of a Asn at -3 and a largealiphatic at -5 or -6. A Pro residue at -2 increases the affinity butappears to be non-essential.

III. Characterization of the PTB Domain of Shc

A comparison of the PTB domains present in Shc and its relativesrevealed the presence of a large number of conserved Arg residues.Several conserved Arg are directly involved in P.Tyr binding by SH2domains. As an initial attempt to characterize PTB domain P.Tyr-binding,all conserved Arg residues in the Shc PTB domain were individuallymutated and GST-fusion proteins containing mutant PTB domains weretested for their ability to bind to the activated NGF receptor (FIG.6A). The three Arg residues and the Lys that are present betweenresidues 97 and 100 were mutated to Met in combination. Of all ten Argresidues tested, only mutation of Arg 175 had a dramatic effect on theaffinity of the Shc PTB domain for the activated NGF receptor (FIG. 6A).Both the Met 175 and the Lys 175 mutants were strongly impaired in theirbinding activity (FIGS. 6A and 6B), indicating that not just a positivecharge but a positive charge in the context of an Arg residue isrequired at this position. The dShc PTB domain contains an Arg atresidue 151, which is homologous to Arg 175 in the human Shc. Wt andmutant dShc PTB domains were tested for their ability to bind to thedrosophila EGF receptor (DER) (FIG. 6B). The ability of Wt and the 175mutant human Shc PTB domains to bind to the human EGF receptor weretested in parallel (FIG. 6B). The Wt dShc PTB domain but not the Arg toAla mutant at position 151 was able to bind efficiently to activated DERin vitro, suggesting that the requirement for the presence of an Argresidue at position 175 in the human Shc PTB domain has been conservedin evolution.

Summary

Shc binding to activated growth factor receptors appears to be animportant step in the initiation of signal transduction towards DNAsynthesis and cell division or differentiation. Shc binding sites areparticularly well characterized in the NGF receptor and in polyomamiddle T antigen. In the NGF receptor Shc binds to Tyr 490 in the juxtamembrane domain (FIG. 3). Mutation of Tyr 490, in addition to mutationof the PLCγ-binding site, completely blocks NGF-induced neuronaldifferentiation in PC12 cells (Stephens et al., 1994). Mutation of Tyr250, which is the Shc binding site, in polyoma middle T antigen blockscellular transformation (Campbell et al., 1994; Dilworth et al., 1994).The EGF receptor also interacts strongly with Shc, although the precisecontribution of different autophosphorylation sites in the EGF receptorcarboxy-terminus remains unresolved (Batzer et al., 1994; Okabayashi etal., 1994).

The PTB domain at the amino-terminus of Shc may be the importantmediator of Shc-growth factor receptor interactions. Asn-Pro-X-P.Tyrmotifs are conserved in a large number of Shc binding proteins andAsn-Pro-X-P.Tyr-containing peptides compete efficiently for Shc PTBbinding to activated growth factor receptors, such as the receptors forEGF and NGF (Blaikie et al., 1994; Campbell et al., 1994; Kavanaugh etal., 1995; van der Geer and Pawson, 1995; van der Geer et al., 1995).Using peptide binding studies with mutant peptides, the presentinventors characterized the nature of the PTB-binding site. The presenceof an Asn residue three residues amino-terminal to the P.Tyr appears tobe absolutely essential for binding to the PTB domain. In contrast, thePro appears to be dispensable for binding to the PTB domain in vitro.Addition of the Pro increases the affinity and this may be important forbinding in vivo, consistent with the observation that the Pro appears tobe conserved in many Shc PTB-binding sites.

It was found that the activated insulin receptor, which also has anautophosphorylation site contained within an Asn-Pro-X-Tyr motif, doesnot bind stably to Shc in vivo or in vitro (Kovacina and Roth, 1993;Pronk et al., 1993). Shc, however, becomes phosphorylated in response toinsulin and the Shc PTB domain was shown to interact with Tyr 960 in theinsulin receptor using the two-hybrid method in yeast (Gustafson et al.,1995). The present inventors have shown that the presence of analiphatic residue five or six residue amino-terminal to the P.Tyr isimportant for high affinity binding by the Shc PTB domain. Aphosphopeptide with two Ala residues at these positions still binds tothe Shc PTB domain but with an affinity that is approximately three foldlower than that for binding of a phosphopeptide with an Ile at eitherposition -6 or -5 (Table 1). The presence of a Ser five residuesamino-terminal to the P.Tyr disrupts high affinity binding completely. Apeptide, derived from the insulin receptor, that lacked the ability tobind to the Shc PTB domain was changed into a PTB-binding site with asingle amino acid substitution at a residue outside the Asn-Pro-X-P.Tyrmotif. Conversely, the ability to bind the Shc PTB domain was destroyedby a single amino acid change outside the Asn-Pro-X-P.Tyr motif in anNGF receptor derived phosphopeptide (Table 1). It appears that differentPTB domains all recognize Asn-Pro-X-P.Tyr or Asn-X-X-P.Tyr and thatfurther specificity results from interactions of the PTB domain withamino acid residues outside this recognition motif. Furthermore, thepresence of particular residues at certain positions within the bindingsite could prohibit certain PTB domains from binding without affectingthe binding of other PTB domains. This is partially illustrated by theobservation that the presence of a Ser five residues amino-terminal tothe P.Tyr prevents binding of the Shc PTB domain. An understanding ofPTB-binding specificity enables accurate predictions to be made as towhich proteins will bind to particular PTB-containing adaptor orsignalling molecules. In addition, it enables manipulation of therepertoire of PTB domain-containing proteins that are recruited bygrowth factor receptors without changing the actual phosphate acceptorsites. For instance, phosphorylation of both the insulin receptorsubstrate 1 (IRS-1) and Shc appears to depend on a low affinityinteraction with the insulin receptor at Tyr 960 (Backer et al., 1990;White et al., 1988; Yonezawa et al., 1994). By changing residuesamino-terminal of the Asn-Pro-X-P.Tyr motif it may be possible toabolish specifically phosphorylation of either one of these polypeptidesby the insulin receptor. Conversely, it may be possible to create aninsulin receptor that interacts much stronger with either Shc or IRS-1.

Several Arg residues that are conserved in SH2 domains have been shownto be directly involved in P.Tyr binding (Pawson, 1995; Pawson and Gish,1992). Based on its functional homology with the SH2 domain furthercharacterization of the PTB domain by mutagenesis of Arg residues thatare conserved in the PTB domains of different members of the Shc familyhas been carried out. The FLVRES sequence (Phe-Leu-Val-Arg-Glu-Ser, SEQID NO:33) has been conserved between SH2 domains with the Arg being theonly invariant residue present in all SH2 domains described thus far(Pawson, 1995; Pawson and Gish, 1992). An Arg residue present within thesequence YLVRYM (Tyr-Leu-Val-Arg-Tyr-Met, SEQ ID NO:34) (residues52-57), possibly representing a rudimentary FLVRES (SEQ ID NO:33) motifin the Shc PTB domain, was mutated without an effect on itsligand-binding abilities; mutation of this residue in SH2 domainsdestroys their ability to bind to phosphotyrosine (Marengere and Pawson,1992; Mayer et al., 1992). This is consistent with the notion that PTBand SH2 domains are structurally unrelated. The studies described hereinhave defmed an Arg residue in the carboxy-terminus of the PTB domainthat is important for its interaction with activated growth factorreceptors. This Arg residue is conserved in dShc and its presence wasfound to be essential for binding of the dShc PTB domain to DER, thedrosophila homolog of the EGF receptor. Thus the need for this Argresidue for PTB-ligand interaction has been conserved in evolutionbetween drosophila and man.

As indicated earlier, Shc appears to be important for signaltransduction downstream of growth factor and cytokine receptors (Burnset al., 1993; Crowe et al., 1994; Cutler et al., 1993; Lanfrancone etal., 1995; Pelicci et al., 1992; Pronk et al., 1993; Ravichandran etal., 1993; Segatto et al., 1993; Yokote et al., 1994). There is evidencethat Shc may be involved in Ras activation presumably through itsinteraction with Grb2 and Sos (Buday and Downward, 1993; Crowe et al.,1994; Egan et al., 1993; Gale et al., 1993; Li et al., 1993; Myers etal., 1994; Rozakis-Adcock et al., 1993; Rozakis-Adcock et al., 1992;Salcini et al., 1994; Sasaoka et al., 1994).

Example 2

The ability of the peptides listed in Table 2 to inhibit the binding ofhuman Shc PTB domains to activated EGF-receptor (R) or NGF-R (Trk) wasinvestigated. The following materials and methods were used in theassays:

Peptides

The peptides are listed in Table 2. In Table 2 the designation "C"refers to a cyclic peptide; C-1,3,4,5 are cyclized by the amino- andcarboxyl termini by an amide bond; C-2 is cyclized by a disulfide bondbetween two cysteines on each of the N- and C-termini; "P" refers topeptides which have penetrating sequences on the N-terminus where P-1and P-2 are basic charged penetrating sequences with the latter havingphosphorylated tyrosine residues; P-3 and P-4 have a hydrophobicpenetrating sequence with the latter having phosphorylated tyrosineresidues; and "P-5" was obtained by coupling with penetratin 1(Appligene) and CGHIIENPQPYFSD (SEQ ID NO:35).

Fusion Proteins

GST-ShcB and GST-R175M fusion proteins were prepared as described in vander Geer et al., 1995.

In vitro binding assay

HER14 cells (3T3 cells expressing EGF-R) were starved in 0.5% CS mediafor 24 hours and stimulated with 100 ng/ml EGF for 5 min. Cells werelysed and mixed with GST, GST-ShcB or GST-R175M beads. Proteins whichbound to beads were resolved on SDS-PAGE and detected byanti-phospho-Tyr antibody (4G10) or by anti-EGF-R.

Results

Inhibition of Binding by Peptides In Vitro

The peptides listed in Table 2 were added to cell lysates at 5 μM,incubated for 30 min., and treated with GST-ShcB beads. Proteins boundto GST-ShcB were detected by anti-phospho-Tyr antibody. A peptide havingthe Shc PTB binding motif of Trk inhibited the association of Shc andEGF-R while a peptide with an amino acid substitution from Asn to Ala(Trk N to A), or an IRS-1 binding site of insulin receptor (Ins) did notinhibit (FIG. 8, Panel A). The peptide designated C-2 inhibited thebinding of Shc and EGF-R completely in vitro (FIG. 8, Panels A, B). P-2,dissolved in Hepes buffer and precipitated, showed weak inhibition (FIG.8, Panel B). The experiment was repeated with the peptides dissolved inDMSO (FIG. 9). P-2 dissolved in DMSO solution showed strong inhibitionat 5 μM; C-2 inhibited complex formation as described previously; andP-1, which is not phosphorylated on Tyr residues did not inhibit theassociation of Shc and EGF-R at 100 μM.

The dose responses of the peptides was investigated using the in vitrobinding assay. While C-2 showed strong inhibition at 5 μM, C-3, C-4, andC-5, did not inhibit (FIG. 10, Panel A). C-1 exhibited about 50%inhibition at 100 μM (FIG. 10, Panel A). P-2 (dissolved in Hepes buffer)at a concentration of 100 μM prevented the association of Shc/EGF-Rcompletely, and it showed slight inhibition at 5 μM compared to thenegative control (P-1) (FIG. 10, Panel B). P-2 dissolved in DMSO showedstrong inhibition at 5 μM while P-2 at 0.5 μM did not block the proteininteraction (FIG. 10, Panel C).

Peptide Localization in Cells

To examine peptide localization, cells were treated with P-1 or P-2peptide for 4 hours, stained with anti-phospho-Tyr andrhodamine-conjugated antibody, and observed with a confocal microscope.A Z-scan was carried out to make images in each 0.15 μm section from thetop of the cells to the bottom. The image analysis of cell stainingdemonstrated that the P-2 peptide localized in the cytoplasm of cells,and not in the nucleus. Cells treated with P-1 peptide were not stainedby anti-phospho-Tyr antibody, confirming the specifity of theimmunofluoroscence staining. In a time course analysis, cells wereserum-starved for 24 hours, and incubated with peptide (5 μM) forvarious time periods. Cells were stained by anti-phosphoro-Tyr antibodyand analyzed under an immunofluoroscence microscope. Cells treated withP-2 peptide retained phospho-peptide for up to 24 hrs. In a doseresponse experiment, cells were starved in serum-free medium for 24hours, cultured with peptide at various concentrations, and stained withanti-phospho-tyrosine antibody (4G10) to detect the peptide containingphospho-tyrosine residue (red) and by Hoechst 33258 for nuclei (blue).P-2 peptide was detected at 0.5 μM and 1 μM in cells . No signals weredetected with P-1 peptide in concentraions up to 1 μM and weaknon-specific staining was observed at 5 μM.

Inhibition of PTB Function in vivo Growth Inhibition of Cells

HER14 cells were starved for 24 hours (FIG. 11, Panel a), or 48 hours(FIG. 11, Panel b), treated with P-1 or P-2 peptide for 2 hrs andstimulated with 100 ng/ml EGF. Cell proliferation was measured by ³H-TdR uptake (FIG. 11). When cells were starved for 24 hrs, P-1 and P-2peptides slightly inhibited the proliferation of cells compared to thepositive control i.e. EGF alone (a). In cells starved for 48 hrs, bothpeptides markedly prevented cell proliferation. In particular, about 84%inhibition was observed at 1.25 μM. Both the P-1 and P-2 peptidedemonstrated inhibitory activity, suggesting a non-specific effect wasinduced by adding peptides. The effect may be due to the internalphosphorylation of P-1 peptide by activated kinase(s) after growthfactor stimulation. The above experiments were repeated with cells whichwere serum starved for 24 hrs. Cells pretreated with P-1 or P-2 did notshow any decrease of cell growth rate when compared to EGF treatedcells. Cells pretreated with C-2 inhibited cell growth roughly in a dosedependent manner (FIG. 12). The experiment was repeated using C-1peptide as a negative control, and C-2 did not inhibit cell growth (FIG.13).

A non-Hodgkin's lymphoma cell line, SupM2 was used in cell proliferationassays as described above. SupM2 has a chromosomal translocation,resulting in the expression of a fusion protein of Alk and Npm. TheAlk/Npm fusion protein has a motif which is expected to be a Shc PTBbinding domain, and the cell proliferation of SupM2 is believed to bedependent on the Shc pathway. SupM2 cells in the indicated cell number(FIG. 14, Panel a) or at a concentration of 2×10⁴ /well (FIG. 14, Panelb), were grown in serum-free RPMI 1640 medium for 24 hr, treated withpeptide for 2 hrs, then, stimulated with 20% FBS overnight, andmonitored by ³ H-tdR pulse. P-1 and P-2 peptides inhibited cell growthto background level (FIG. 14), and cell proliferation was completelyinhibited at 70 nM. To examine the optimum dose of peptides, serialdilutions of peptides were used in the assay. At 60 pM, both peptidessuppressed the cell growth completely.

Inhibition of PTB Function in vivo. MAPK Activation

The phosphorylation state of MAPK was examined after treatment with thepeptides. PC12 cells were treated with peptide, stimulated with 50 ng/mlNGF, and a cell lysate was prepared. Proteins were resolved onacrylamide gel to separate phosphorylated- and non-phosphorylated MAPK.Erk-1 was detected by Western blotting. FIG. 15, Panel a shows theseries of samples treated with P-1 or P-2, and Panel b shows the samplestreated with C-1 or C-2. When cells were treated with NGF, the bandsexpected for Erk-1 and Erk-2 were slightly shifted and showed highermolecular weights, demonstrating phosphorylation of Erk-1 and Erk-2.However, no inhibition was detected in the group treated with P-2 orC-2. To confirm this result, cell lysates of PC12 described above wereimmunoprecipitated with anti-Erk-1 antibody, and the proteins weredetected by anti-phospho-Tyr antibody (FIG. 16). In FIG. 16, Panel ashows samples treated with P-1 or P-2, and Panel b shows samples treatedwith C-1 or C-2. Two major bands were detected with predicted molecularweights of about 47 kDa and about 42 kDa, respectively. A weak band of47 kDa was detected in a sample treated with preimmune serum. Therefore,pp47 appeared to be a non-specific protein whereas pp42 isphosphorylated Erk-1. No differences in phosphorylation state of Erk-1were observed among the groups treated with the peptides.

The above described experiment was repeated using 3T3Trk cellsstimulated with NGF. The results were similar to that obtained with PC12cells. No specific inhibition of Erk-1 and Erk-2 phosphorylation by C-2or P-2 was observed.

In summary, the efficacy of the peptides in Table 2 were assayed for theability to inhibit the interaction between the Shc PTB domain and growthfactor receptors. P-2 and C-2 peptides demonstrated strong inhibitoryactivities in in vitro binding assays. P-2 was found to localize in thecell cytoplasm by simply adding the peptide into the culture medium. Inpreliminary experiments, specific inhibition of cell growth or of MAPKactivation by the peptides was not demonstrated in vivo. However, the invivo assay system requires further optimization.

Having illustrated and described the principles of the invention in apreferred embodiment, it should be appreciated to those skilled in theart that the invention can be modified in arrangement and detail withoutdeparture from such principles. We claim all modifications coming withinthe scope of the following claims.

All publications, patents and patent applications referred to herein areincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

Below full citations are set out for the references referred to in thespecification is a listing and detailed legends for the figures areprovided.

The application contains sequence listings which form part of theapplication.

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Detailed Figure Legends

FIG. 1A and FIG. 1B. The Asn present within the Asn-Pro-X-P.Tyr motif isessential for binding to the PTB domain. FIG. 1A. GST (lane 2) and GSTShc PTB (lanes 1, 3-6) fusion proteins bound to glutathione-agarose wereincubated with NGF receptors present in lysates from control (lane 1)and NGF-stimulated (lanes 2-7) cells in the absence (lane 1-3) andpresence of Wt (lane 4) and mutant (lanes 5 and 6) competing P.Tyrcontaining peptides, based on the sequence around Tyr 490 the Shc PTBdomain binding site in the NGF receptor. Bound proteins were analyzed byanti-P.Tyr immunoblotting. Competing peptides, Wt NGF receptor (WtNGFR): His-Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp (SEQ ID NO:1) (lane4); NGF receptor Asn(-3)Ala (NGFR Ala -3) mutant:His-Ile-Ile-Glu-Ala-Pro-Gln-P.Tyr-Phe-Ser-Asp (SEQ ID NO:37) (lane 5);NGF receptor Pro(-2)Ala (NGFR Pro -2):His-Ile-Ile-Glu-Asn-Ala-Gln-P.Tyr-Phe-Ser-Asp (amino acids 1-11 of SEQID NO:38) (lane 6). FIG. 1B. The blot shown under A was stripped andreprobed with an antiserum raised against the NGF receptor.

FIG. 2. Substitution of either the Asn or the Pro in the PTB-bindingsite affects its ability to bind to the PTB domain. Surface plasmonresonance technology was used to test the ability of Wt and mutantphosphopeptides, based on the sequence around Tyr 490 the Shc-bindingsite in the NGF receptor for their ability to compete for binding of theGst-Shc PTB domain fusion protein to the immobilized polyoma middle Tantigen peptide (L-S-L-L-S-N-P-T-P.Y-S-V-M-R-S-K, SEQ ID NO:36). Wt.H-I-I-E-N-P-Q-P.Y-F-S-D: (SEQ ID NO:1): (A); Ala -3,H-I-I-E-A-P-Q-P.Y-F-S-D (SEQ ID NO:37): (o); Ala -2,H-I-I-E-N-A-Q-P.Y-F-S-DP (SEQ ID NO:38) ().

FIG. 3. Presence of a Asn-Pro-X-P.Tyr motif in the juxta membranedomains of the NGF and insulin receptors. Both the NGF receptor and theinsulin receptor contain an autophosphorylation site within anAsn-Pro-X-P.Tyr motif in the juxta membrane domain, between the membraneand the kinase domain. In both receptors the tyrosine residues withinthese motif become phosphorylated upon receptor activation, but incontrast to the NGF receptor, the insulin receptor lacks the ability tostably associate with Shc.

FIG. 4A and FIG. 4B. The Shc PTB domain does not stably bind to theAsn-Pro-X-P.Tyr motif in the insulin receptor. FIG. 4A. Anti-Shcimmunoprecipitates (lanes 1, 2, 5, 6, 9, and 10) from control (lanes 1,5, and 9) and growth factor-stimulated (lanes 2, 6, and 10) NIH3T3fibroblasts expressing Wt (lanes 1 and 2; NGFR) or Phe 490 mutant (lanes5 and 6; F490NGFR) NGF receptors, or CHO cells expressing Wt insulinreceptors (lanes 9 and 10; IR) were analyzed by anti-P.Tyrimmunoblotting. Anti-NGF receptor (lanes 3, 4, 7, and 8) andanti-insulin receptor immunoprecipitates (lanes 11 and 12) from control(lanes 3, 7, and 11) and growth factor stimulated (lanes 4, 8, and 12)were analyzed in parallel. FIG. 4B. Wt (lanes 1 and 2) and Phe 490mutant (5 and 6) NGF receptors present in lysates from control (lanes 1and 5) and NGF-stimulated (lanes 2 and 6) cells expressing Wt (NGFR) orPhe 490 mutant (F490NGFR) and insulin receptors (IR) present in lysatesfrom control (lane 9) and insulin-stimulated (lane 10) cells wereincubated with GST-Shc PTB fusion proteins bound to glutathione-agarose.Bound proteins were analyzed by anti-P.Tyr immunoblotting. Anti-NGFreceptor immunoprecipitates (lanes 3, 4, 7, and 8) and anti-insulinreceptor immunoprecipitates (lanes 11 and 12) from control (lanes 3, 7,and 11) and growth factor-stimulated (lanes 4, 8, and 12) cells wereanalyzed in parallel.

FIG. 5A and FIG. 5B. An aliphatic residue five or six amino acidsamino-terminal to the P.Tyr is an important determinant for Shc PTBbinding. FIG. 5A. GST-Shc PTB domain fusion proteins bound toglutathione-agarose were incubated with activated NGF receptors presentin lysates of NGF-stimulated cells in the absence (lane 1) or presence(lanes 2-7) of 2 μM competing Wt and mutant phosphotyrosine containingpeptides based on the sequence around Tyr 490, the Shc PTB domainbinding site in the NGF receptor (lanes 2-5) or Tyr 960 anautophosphorylation site present within an Asn-Pro-X-P.Tyr motif in theinsulin receptor (lanes 6 and 7). Wt NGF receptor peptide (Wt-NGFR, lane2): H-I-I-E-N-P-Q-p.Y-F-S-D SEQ ID NO:39; Ala-6 NGF receptor peptide(NGFR-HAI): H-A-I-E-N-P-Q-p.Y-F-S-D (SEQ ID NO:2); Ala-6, Ala-5 NGFreceptor peptide (NGFR-HAA): H-A-A-E-N-P-Q-p.Y-F-S-D (SEQ ID NO:40);Ala-6, Ser-5 NGF receptor peptide (NGFR-HAS): H-A-S-E-N-P-Q-p.Y-F-S-D(SEQ ID NO:41); Wt insulin receptor peptide (Wt-IR):Y-A-S-S-N-P-E-p.Y-L-S-A (SEQ ID NO:42); Ile-5 insulin receptor peptide(IR-YAI): Y-A-I-S-N-P-E-p.Y-L-S-A (SEQ ID NO:5). Bound proteins wereanalyzed by P.Tyr. blotting. FIG. 5B. Phosphopeptides based on thesequence around Tyr 490, the Shc-binding site in the NGF receptor(H-I-I-E-N-P-Q-p.Y-F-S-D, SEQ ID NO:5 ()) or Tyr 960 in the insulinreceptor (Y-A-S-S-N-P-E-p.Y-L-S-A (SEQ ID NO:42) (o)) and substitutionsat position -5 and -6 with respect to the P.Tyr in the NGF receptorpeptides (H-A-S-E-N-P-Q-p.Y-F-S-D (SEQ ID NO:41) (▪)) and the insulinreceptor peptide (Y-A-I-S-N-P-E-p.Y-L-S-A SEQ ID NO:5 (▴)) were testedby surface plasmon resonance analysis technology for their ability tocompete for the binding of the GST-Shc PTB domain to the immobilizedpolyoma middle T antigen peptide (L-S-L-L-S-N-P-T-p.Y-S-V-M-R-S-K, SEQID NO:36).

FIG. 6A and FIG. 6B. The requirement for an Arg residue at position 175in the human Shc PTB domain has been conserved in evolution. FIG. 6A.GST fusion proteins containing Wt (lanes 1 and 2) or mutant (lanes 3-11)Shc PTB domains were incubated with NGF receptors present in lysates ofcontrol (lane 1) and NGF-stimulated cells (lanes 2-11). Bound proteinswere analyzed by anti-P.Tyr blotting. FIG. 6B. Human EGF receptors boundto GST fusion proteins containing Wt (lanes 1 and 2) or Met 175 (lane 3)and Lys 175 (lane 4) mutant human Shc PTB domains in lysates fromcontrol (lane 1) or EGF-stimulated cells (lanes 2-4) were analyzed byanti-P.Tyr blotting. In parallel GST (lane 8) and GST fusion proteinscontaining Wt (lane 7) or an Ala 151 mutant (lane 9) drosophila Shc PTBdomain bound to glutathione-agarose, were incubated with fly lysatescontaining activated Torso-DER chimeric proteins that contain thecytoplasmic domain of DER; bound proteins were detected by anti-P.Tyrblotting. An anti-Shc (lane 5) and a normal rabbit serumimmunoprecipitate (lane 6) from the same fly lysates are shown ascontrols.

FIG. 8. Peptides Competition in In Vitro Binding Assay. Cell Lysateswere preincubated with 5 μM of appropriate peptides for 30 min. at 4° C.Then, proteins were precipitated by each binder, resolved on SDA-PAGE.Detection was carried out by anti-phospho-tyrosine antibody. Ins.:IRS-1binding domain on insulin-R.

FIG. 9. Competition Assay of Penetrating Peptide. Cell lysates werepre-treated with appropriate peptides. Proteins were precipitated byGST-ShcB and detected by anti-phospho-tyrosine antibody. Peptides wereprepared in DMSO solution.

FIG. 10. Dose-Response Analysis of Peptides in in vitro Binding Assay.Cell Lysates were prepared and incubated with appropriate peptides invarious concentrations. Proteins were precipitated by GST-ShcB andresolved on 10% SDS-PAGE gel. Anti-phospho-tyrosine antibody was usedfor detection. P-1 and P-2 peptides were dissolved in Hepes buffer (B),and in DMSO solution (C).

FIG. 11. Proliferation of HER14 cells treated with peptides. Cells werestarved for 24 (a) or 48 hrs (b) prior to stimulation. Cells weretreated with appropriate peptide for 2 hrs, then stimulated with 100ng/ml EGF overnight. Cell proliferation was monitored by ³ H-TdR uptake.

FIG. 12. Proliferation of HER14 cells. HER14 cells were cultured inserum-free D-MEM medium in 96-well plates for 24 hrs. Appropriatepeptides were added in various concentrations 2 hrs prior to EGFstimulation. Cell proliferation was induced with 100 ng/ml EGFovernight, then monitored by ³ H-TdR uptake.

FIG. 13. Proliferation of HIER14 cells treated with cyclic peptides.Cells were starved for 48 hrs in serum-free D-MEM medium. Appropriatepeptide was added in various concentrations and cultured for 2 hrs priorto EGF stimulation. Cell proliferation was induced with 100 ng/ml EGFovernight and monitored by ³ H-TdR uptake.

FIG. 14. Proliferation of SupM2 cells treated with peptides. SupM2 cellswere starved in serum-free RPMI 1640 medium for 24 hrs in the indicatedcell number (a) or 2×10⁴ /well (b). Cells were treated with penetratingpeptide in various concentrations for 2 hrs prior to cell stimulation.Cell proliferation was induced with 20% FPS overnight and monitored by ³H-TdR pulse.

FIG. 15. MAPK Activation on PC12 Cells Treated with Peptides. PC 12cells were treated with appropriate peptide at various concentrations.Cells were stimulated with 50 ng/ml NGF for 5 min., then cell lysateswere prepared by standard methods. Each lane contains 10 μg protein andMAPK (Erk-1) was detected by anti-Erk-1 polyclonal Ab. Arrows representactivated Erk-1/2 (b).

FIG. 16. Detection of Activated MAPK on PC12 Cells Treated withPeptides. PC12 cells were treated with peptides for 4 hrs prior tostimulation. Cells were stimulated with 50 ng/ml NGF for 5 min. and celllysates were prepared according to standard methods. 500 μg of celllysates were immunoprecipitated with anti-Erk-1 polyclonal antibody, andactivated Erk-1 proteins were detected by Western blotting ofanti-phospho-tyrosine antibody (4G10). Arrow represents activated(phosphorylated) Erk-1 and asterisk shows non-specific band.

                  TABLE 1                                                         ______________________________________                                        Phosphotyrosine-containing peptides                                                                    IC 50                                                ______________________________________                                        H-I-I-E-N-P-Q-P.Y-F-S-D  175 nM                                               H-I-I-E-A-P-Q-P.Y-F-S-D               80000 nM                                H-I-I-E-N-A-Q-P.Y-F-S-D                2500 nM                                H-A-I-E-N-P-Q-P.Y-F-S-D                  20 nM                                H-I-A-E-N-P-Q-P.Y-F-S-D                 250 nM                                H-A-A-E-N-P-Q-P.Y-F-S-D                 475 nM                                H-A-S-E-N-P-Q-P.Y-F-S-D               15000 nM                                Y-A-S-S-N-P-E-P.Y-L-S-A                7000 nM                                Y-A-I-S-N-P-E-P.Y-L-S-A                  90 nM                                ______________________________________                                         Table 1. Peptide competition of the GSTShc FTB domain binding to a polyom     middle T antigen phosphopeptide (LS-L-L-S-N-P-T-P.Y SV-M-R-S-K, SEQ ID NO     36). Surface plasmon resonance technology was used to evaluate the abilit     of phosphopeptides derived from sequences around the Tyr 490, the Shc         binding site in the NGF receptor (HI-I-E-N-P-Q-P.Y FS-D, SEQ ID NO:1) and     Tyr 960 in the insulin receptor (YA-S-S-N-P-P.Y LS-A, SEQ ID NO: 42) to       bind  # to the Shc PTB domain. Amino acid substitutions were introduced       into the peptides (shown in bold). Peptide concentrations that inhibit        ending by 50% (IC.sub.50) are listed. The SEQ ID NOs. for the peptides        from the top to the bottom sequence in the Table are ID Nos. 1, 37, amino     acids 1-11 of SEQ ID NO: 38, 43, 3, 40, 41, 42, and 44, respectively.    

                  TABLE 2                                                         ______________________________________                                        Code     Amino acid sequence                                                  ______________________________________                                        Trk wild type                                                                          HIIENPQpYFSD                                                                              175 nM                                                            IENPQpYFSD   4 μM                                                 C-1      Cyclo-(IENPQpYFSPG)                                                  C-2                                                                                     ##STR1##                                                            C-3      Cyclo-(NPQpY)                                                        C-4      Cyclo-(NPQpYG)                                                       C-5      Cyclo-(NPQpYGG)                                                      P-1      RQIKIWFQNRRMKWKK-HIIENPQYFSD                                         P-2      RQIKIWFQNRRMKWKK-HIIENPQpYFSD                                        P-3      Biotin-ACA-AAVALLPAVLLALLAP-HIIENPQYFSD                              P-4      Biotin-ACA-AAVALLPAVLLALLAP-HIIENPQpYFSD                             P-5                                                                                     ##STR2##                                                                     (RQIKIWFQNRRMKWKK?,                                                           amino acids 1-16 of SEQ ID NO:51)                                    ______________________________________                                         The SEQ ID NOs. for the Trk wild type peptides are ID. Nos. 1 and 45, and     the SEQ ID NOs for C1, C2, C3, C4, C5, P1, P2, P3, P4, and P5 are ID. Nos     46, 47, 48, 49, 50, 51, 52, 53, 54, 55, respectively.                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 60                                              - <210> SEQ ID NO 1                                                           <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 1                                                           - His Ile Ile Glu Asn Pro Gln Tyr Phe Ser As - #p                             #                 10                                                          - <210> SEQ ID NO 2                                                           <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 2                                                           - His Ala Ile Glu Asn Pro Gln Tyr Phe Ser As - #p                             #                 10                                                          - <210> SEQ ID NO 3                                                           <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 3                                                           - His Ile Ala Glu Asn Pro Gln Tyr Phe Ser As - #p                             #                 10                                                          - <210> SEQ ID NO 4                                                           <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (7)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 4                                                           - Ile Ile Glu Asn Pro Gln Tyr Phe Ser Asp Al - #a                             #                 10                                                          - <210> SEQ ID NO 5                                                           <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           #position number 8ATION: Phosphorylated at Tyr in                             - <400> SEQUENCE: 5                                                           - Tyr Ala Ile Ser Asn Pro Glu Tyr Leu Ser Al - #a                             #                 10                                                          - <210> SEQ ID NO 6                                                           <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 6                                                           - Thr Trp Ile Glu Asn Lys Leu Tyr Gly Met Se - #r Asp                         #                 10                                                          - <210> SEQ ID NO 7                                                           <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 7                                                           - Thr Trp Ile Glu Asn Lys Leu Tyr Gly Thr Se - #r Asp                         #                 10                                                          - <210> SEQ ID NO 8                                                           <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 8                                                           - Leu Leu Leu Ser Asn Pro Ala Tyr Arg Leu Le - #u Leu                         #                 10                                                          - <210> SEQ ID NO 9                                                           <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           #position number 8ATION: Phosphorylated at Tyr in                             - <400> SEQUENCE: 9                                                           - Tyr Ala Ser Ser Asn Pro Glu Tyr Leu Ser Al - #a Ser                         #                 10                                                          - <210> SEQ ID NO 10                                                          <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 10                                                          - Val Ser Val Asp Asn Pro Glu Tyr Leu Leu As - #n Ala                         #                 10                                                          - <210> SEQ ID NO 11                                                          <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 11                                                          - Ser Leu Leu Ser Asn Pro Thr Tyr Ser Val Me - #t Arg                         #                 10                                                          - <210> SEQ ID NO 12                                                          <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 12                                                          - Asn Glu Met Ile Asn Pro Asn Tyr Ile Gly Me - #t Gly                         #                 10                                                          - <210> SEQ ID NO 13                                                          <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 13                                                          - Glu Met Phe Glu Asn Pro Leu Tyr Gly Ser Va - #l Ser                         #                 10                                                          - <210> SEQ ID NO 14                                                          <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (6)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 14                                                          - Ile Glu Asn Pro Gln Tyr Phe                                                   1               5                                                           - <210> SEQ ID NO 15                                                          <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (6)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 15                                                          - Ile Glu Asn Pro Gln Tyr Phe Ser                                               1               5                                                           - <210> SEQ ID NO 16                                                          <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (6)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 16                                                          - Ala Glu Asn Pro Gln Tyr Phe                                                   1               5                                                           - <210> SEQ ID NO 17                                                          <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (6)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 17                                                          - Ile Glu Asn Pro Gln Tyr Phe Ser Pro Gly                                     #                 10                                                          - <210> SEQ ID NO 18                                                          <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (6)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 18                                                          - Ile Ser Asn Pro Glu Tyr Leu                                                   1               5                                                           - <210> SEQ ID NO 19                                                          <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 19                                                          - Val Leu Ala Asp Asn Pro Ala Tyr Arg Ser Al - #a                             #                 10                                                          - <210> SEQ ID NO 20                                                          <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (9)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 20                                                          - Ala Leu Leu Leu Ser Asn Pro Ala Tyr Arg Le - #u Leu Leu Ala                 #                 10                                                          - <210> SEQ ID NO 21                                                          <211> LENGTH: 18                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (11)                                                          #position number 11TION: Phosphorylated at Tyr in                             - <400> SEQUENCE: 21                                                          - Gly Pro Leu Tyr Ala Ser Ser Asn Pro Glu Ty - #r Leu Ser Ala Ser Asp         #                 15                                                          - Val Phe                                                                     - <210> SEQ ID NO 22                                                          <211> LENGTH: 15                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (9)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 22                                                          - Pro Val Ser Val Asp Asn Pro Glu Tyr Leu Le - #u Asn Ala Gln Lys             #                 15                                                          - <210> SEQ ID NO 23                                                          <211> LENGTH: 15                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (9)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 23                                                          - Leu Ser Leu Leu Ser Asn Pro Thr Tyr Ser Va - #l Met Arg Ser Lys             #                 15                                                          - <210> SEQ ID NO 24                                                          <211> LENGTH: 18                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (12)                                                          <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 24                                                          - Val Ser Ser Leu Asn Glu Met Ile Asn Pro As - #n Tyr Ile Gly Met Gly         #                 15                                                          Pro Phe                                                                       - <210> SEQ ID NO 25                                                          <211> LENGTH: 20                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (14)                                                          <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 25                                                          - Leu Leu Leu Thr Lys Pro Glu Met Phe Glu As - #n Pro Leu Tyr Gly Ser         #                 15                                                          - Val Ser Ser Phe                                                                          20                                                               - <210> SEQ ID NO 26                                                          <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (6)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                <220> FEATURE:                                                                <221> NAME/KEY: PEPTIDE                                                       <222> LOCATION: (1)..(10)                                                      cyclicTHER INFORMATION: other                                                - <400> SEQUENCE: 26                                                          - Ile Glu Asn Pro Gln Tyr Phe Ser Pro Gly                                     #                 10                                                          - <210> SEQ ID NO 27                                                          <211> LENGTH: 13                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (7)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                <220> FEATURE:                                                                <221> NAME/KEY: PEPTIDE                                                       <222> LOCATION: (1)..(13)                                                      cyclicTHER INFORMATION: other                                                - <400> SEQUENCE: 27                                                          - Ile Ile Glu Asn Pro Gln Tyr Phe Ser Asp Al - #a Pro Gly                     #                 10                                                          - <210> SEQ ID NO 28                                                          <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                <220> FEATURE:                                                                <221> NAME/KEY: PEPTIDE                                                       <222> LOCATION: (1)..(14)                                                      cyclicTHER INFORMATION: other                                                - <400> SEQUENCE: 28                                                          - His Ile Ile Glu Asn Pro Gln Tyr Phe Ser As - #p Ala Pro Gly                 #                 10                                                          - <210> SEQ ID NO 29                                                          <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                <220> FEATURE:                                                                <221> NAME/KEY: PEPTIDE                                                       <222> LOCATION: (1)..(10)                                                      cyclicTHER INFORMATION: other                                                - <400> SEQUENCE: 29                                                          - Cys Ile Ile Glu Asn Pro Gln Tyr Phe Cys                                     #                 10                                                          - <210> SEQ ID NO 30                                                          <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 30                                                          - Tyr Ala Ser Ser Asn Pro Glu Tyr Leu Ser Al - #a                             #                 10                                                          - <210> SEQ ID NO 31                                                          <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (9)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 31                                                          - His Ala Ser Glu Asn Pro Gln Tyr Phe Ser As - #p                             #                 10                                                          - <210> SEQ ID NO 32                                                          <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (4)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 32                                                          - Asn Pro Glu Tyr                                                               1                                                                           - <210> SEQ ID NO 33                                                          <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                - <400> SEQUENCE: 33                                                          - Phe Leu Val Arg Glu Ser                                                       1               5                                                           - <210> SEQ ID NO 34                                                          <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                - <400> SEQUENCE: 34                                                          - Tyr Leu Val Arg Tyr Met                                                       1               5                                                           - <210> SEQ ID NO 35                                                          <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                - <400> SEQUENCE: 35                                                          - Cys Gly His Ile Ile Glu Asn Pro Gln Pro Ty - #r Phe Ser Asp                 #                 10                                                          - <210> SEQ ID NO 36                                                          <211> LENGTH: 15                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (9)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 36                                                          - Leu Ser Leu Leu Ser Asn Pro Thr Tyr Ser Va - #l Met Arg Ser Lys             #                 15                                                          - <210> SEQ ID NO 37                                                          <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 37                                                          - His Ile Ile Glu Ala Pro Gln Tyr Phe Ser As - #p                             #                 10                                                          - <210> SEQ ID NO 38                                                          <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 38                                                          - His Ile Ile Glu Asn Ala Gln Tyr Phe Ser As - #p Pro                         #                 10                                                          - <210> SEQ ID NO 39                                                          <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 39                                                          - His Ile Ile Glu Asn Pro Gln Tyr Phe Ser As - #p                             #                 10                                                          - <210> SEQ ID NO 40                                                          <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 40                                                          - His Ala Ala Glu Asn Pro Gln Tyr Phe Ser As - #p                             #                 10                                                          - <210> SEQ ID NO 41                                                          <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 41                                                          - His Ala Ser Glu Asn Pro Gln Tyr Phe Ser As - #p                             #                 10                                                          - <210> SEQ ID NO 42                                                          <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           #position number 8ATION: Phosphorylated at Tyr in                             - <400> SEQUENCE: 42                                                          - Tyr Ala Ser Ser Asn Pro Glu Tyr Leu Ser Al - #a                             #                 10                                                          - <210> SEQ ID NO 43                                                          <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 43                                                          - His Ala Ile Glu Asn Pro Gln Tyr Phe Ser As - #p                             #                 10                                                          - <210> SEQ ID NO 44                                                          <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           #position number 8ATION: Phosphorylated at Tyr in                             - <400> SEQUENCE: 44                                                          - Tyr Ala Ile Ser Asn Pro Glu Tyr Leu Ser Al - #a                             #                 10                                                          - <210> SEQ ID NO 45                                                          <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (6)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 45                                                          - Ile Glu Asn Pro Gln Tyr Phe Ser Asp                                           1               5                                                           - <210> SEQ ID NO 46                                                          <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (6)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                <220> FEATURE:                                                                <221> NAME/KEY: PEPTIDE                                                       <222> LOCATION: (1)..(10)                                                      cyclicTHER INFORMATION: other                                                - <400> SEQUENCE: 46                                                          - Ile Glu Asn Pro Gln Tyr Phe Ser Pro Gly                                     #                 10                                                          - <210> SEQ ID NO 47                                                          <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                <220> FEATURE:                                                                <221> NAME/KEY: PEPTIDE                                                       <222> LOCATION: (1)..(10)                                                      cyclicTHER INFORMATION: other                                                - <400> SEQUENCE: 47                                                          - Cys Ile Ile Glu Asn Pro Gln Tyr Phe Cys                                     #                 10                                                          - <210> SEQ ID NO 48                                                          <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (4)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                <220> FEATURE:                                                                <221> NAME/KEY: PEPTIDE                                                       <222> LOCATION: (1)..(4)                                                      <223> OTHER INFORMATION: cyclic                                               - <400> SEQUENCE: 48                                                          - Asn Pro Gln Tyr                                                               1                                                                           - <210> SEQ ID NO 49                                                          <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (4)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                <220> FEATURE:                                                                <221> NAME/KEY: PEPTIDE                                                       <222> LOCATION: (1)..(5)                                                      <223> OTHER INFORMATION: cyclic                                               - <400> SEQUENCE: 49                                                          - Asn Pro Gln Tyr Gly                                                           1               5                                                           - <210> SEQ ID NO 50                                                          <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (4)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                <220> FEATURE:                                                                <221> NAME/KEY: PEPTIDE                                                       <222> LOCATION: (1)..(6)                                                      <223> OTHER INFORMATION: cyclic                                               - <400> SEQUENCE: 50                                                          - Asn Pro Gln Tyr Gly Gly                                                       1               5                                                           - <210> SEQ ID NO 51                                                          <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                - <400> SEQUENCE: 51                                                          - Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Ar - #g Met Lys Trp Lys Lys         #                 15                                                          - His Ile Ile Glu Asn Pro Gln Tyr Phe Ser As - #p                             #             25                                                              - <210> SEQ ID NO 52                                                          <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (24)                                                          <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 52                                                          - Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Ar - #g Met Lys Trp Lys Lys         #                 15                                                          - His Ile Ile Glu Asn Pro Gln Tyr Phe Ser As - #p                             #             25                                                              - <210> SEQ ID NO 53                                                          <211> LENGTH: 30                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                - <400> SEQUENCE: 53                                                          - Ala Cys Ala Ala Ala Val Ala Leu Leu Pro Al - #a Val Leu Leu Ala Leu         #                 15                                                          - Leu Ala Pro His Ile Ile Glu Asn Pro Gln Ty - #r Phe Ser Asp                 #             30                                                              - <210> SEQ ID NO 54                                                          <211> LENGTH: 30                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (27)                                                          <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 54                                                          - Ala Cys Ala Ala Ala Val Ala Leu Leu Pro Al - #a Val Leu Leu Ala Leu         #                 15                                                          - Leu Ala Pro His Ile Ile Glu Asn Pro Gln Ty - #r Phe Ser Asp                 #             30                                                              - <210> SEQ ID NO 55                                                          <211> LENGTH: 15                                                              <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (12)                                                          <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 55                                                          - Ser Ser Cys Gly His Ile Ile Glu Asn Pro Gl - #n Tyr Phe Ser Asp             #                 15                                                          - <210> SEQ ID NO 56                                                          <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Phosphorylated at Tyr                                - <400> SEQUENCE: 56                                                          - His Ile Ile Glu Asn Pro Gln Tyr                                               1               5                                                           - <210> SEQ ID NO 57                                                          <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (8)                                                           #position 8 INFORMATION: Phosphorylated at Tyr in                             - <400> SEQUENCE: 57                                                          - Tyr Ala Ser Ser Asn Pro Glu Tyr                                               1               5                                                           - <210> SEQ ID NO 58                                                          <211> LENGTH: 307                                                             <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                - <400> SEQUENCE: 58                                                          - Met Asn Lys Leu Ser Gly Gly Gly Gly Arg Ar - #g Thr Arg Val Glu Gly         #                 15                                                          - Gly Gln Leu Gly Gly Glu Glu Trp Ile Arg Hi - #s Gly Ser Phe Val Asn         #             30                                                              - Lys Pro Thr Arg Gly Trp Leu His Pro Asn As - #p Lys Val Met Gly Pro         #         45                                                                  - Gly Val Ser Tyr Leu Val Arg Tyr Met Gly Cy - #s Val Glu Val Leu Gln         #     60                                                                      - Ser Met Arg Ala Leu Asp Phe Asn Ile Arg Th - #r Gln Val Thr Arg Glu         # 80                                                                          - Ala Ile Ser Leu Val Cys Glu Ala Val Pro Gl - #y Ala Lys Gly Ala Thr         #                 95                                                          - Arg Arg Arg Lys Pro Cys Ser Arg Pro Leu Se - #r Ser Ile Leu Gly Arg         #           110                                                               - Ser Asn Leu Lys Phe Ala Gly Met Pro Ile Th - #r Leu Thr Val Ser Thr         #       125                                                                   - Ser Ser Leu Asn Leu Met Ala Ala Asp Cys Ly - #s Gln Ile Ile Ala Asn         #   140                                                                       - His His Met Gln Ser Ile Ser Phe Ala Ser Gl - #y Gly Asp Pro Asp Thr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ala Glu Tyr Val Ala Tyr Val Ala Lys Asp Pr - #o Val Asn Gln Arg Ala         #               175                                                           - Cys His Ile Leu Glu Cys Pro Glu Gly Leu Al - #a Gln Asp Val Ile Ser         #           190                                                               - Thr Ile Gly Gln Ala Phe Glu Leu Arg Phe Ly - #s Gln Tyr Leu Arg Asn         #       205                                                                   - Pro Pro Lys Leu Val Thr Pro His Asp Arg Me - #t Ala Gly Phe Asp Gly         #   220                                                                       - Ser Ala Trp Asp Glu Glu Glu Glu Glu Pro Pr - #o Asp His Gln Tyr Tyr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asn Asp Phe Pro Gly Lys Glu Pro Pro Leu Gl - #y Gly Val Val Asp Met         #               255                                                           - Arg Leu Arg Glu Gly Ala Ala Pro Gly Ala Al - #a Arg Pro Thr Ala Pro         #           270                                                               - Asn Ala Gln Thr Pro Ser His Leu Gly Ala Th - #r Leu Pro Val Gly Gln         #       285                                                                   - Pro Val Gly Gly Asp Pro Glu Val Arg Lys Gl - #n Met Pro Pro Pro Pro         #   300                                                                       - Pro Cys Pro                                                                 305                                                                           - <210> SEQ ID NO 59                                                          <211> LENGTH: 303                                                             <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                - <400> SEQUENCE: 59                                                          - Met Asn Lys Leu Ser Gly Gly Gly Gly Arg Ar - #g Thr Arg Val Glu Gly         #                 15                                                          - Gly Gln Leu Gly Gly Glu Glu Trp Ile Arg Hi - #s Gly Ser Phe Val Asn         #             30                                                              - Lys Pro Thr Arg Gly Trp Leu His Pro Asn As - #p Lys Val Met Gly Pro         #         45                                                                  - Gly Val Ser Tyr Leu Val Arg Tyr Met Gly Cy - #s Val Glu Val Leu Gln         #     60                                                                      - Ser Met Arg Ala Leu Asp Phe Asn Ile Arg Th - #r Gln Val Thr Arg Glu         # 80                                                                          - Ala Ile Ser Leu Val Cys Glu Ala Val Pro Gl - #y Ala Lys Gly Ala Thr         #                 95                                                          - Arg Arg Arg Lys Pro Cys Ser Arg Pro Leu Se - #r Ser Ile Leu Gly Arg         #           110                                                               - Ser Asn Leu Lys Phe Ala Gly Met Pro Ile Th - #r Leu Thr Val Ser Thr         #       125                                                                   - Ser Ser Leu Asn Leu Met Ala Ala Asp Cys Ly - #s Gln Ile Ile Ala Asn         #   140                                                                       - His His Met Gln Ser Ile Ser Phe Ala Ser Gl - #y Gly Asp Pro Asp Thr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ala Glu Tyr Val Ala Tyr Val Ala Lys Asp Pr - #o Val Asn Gln Arg Ala         #               175                                                           - Cys His Ile Leu Glu Cys Pro Glu Gly Leu Al - #a Gln Asp Val Ile Ser         #           190                                                               - Thr Ile Gly Gln Ala Phe Glu Leu Arg Phe Ly - #s Gln Tyr Leu Arg Asn         #       205                                                                   - Pro Pro Lys Leu Val Thr Pro His Asp Arg Me - #t Ala Gly Phe Asp Gly         #   220                                                                       - Ser Ala Trp Asp Glu Glu Glu Glu Glu Pro Pr - #o Asp His Gln Tyr Tyr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asn Asp Phe Pro Gly Lys Glu Pro Pro Leu Gl - #y Gly Val Val Asp Met         #               255                                                           - Arg Leu Arg Glu Gly Ala Ala Arg Pro Thr Le - #u Pro Ser Ala Gln Met         #           270                                                               - Ser Ser His Leu Gly Ala Thr Leu Pro Ile Gl - #y Gln His Ala Ala Gly         #       285                                                                   - Asp His Glu Val Arg Lys Gln Met Phe Leu Pr - #o Pro Pro Cys Pro             #   300                                                                       - <210> SEQ ID NO 60                                                          <211> LENGTH: 259                                                             <212> TYPE: PRT                                                               <213> ORGANISM: phosphotyrosine binding domain                                - <400> SEQUENCE: 60                                                          - Met Pro Lys Asn Gly Asp Ala Gly Gly Arg Se - #r Gly Ser Gly Thr Ile         #                 15                                                          - Ser Asp Gly Cys Ile Tyr Pro Asp Asp Val Il - #e Met Gly Val Gly Val         #             30                                                              - Ala Phe Asn Val Arg Tyr Thr Gly Cys Val Gl - #u Val Lys Thr Ser Met         #         45                                                                  - Lys Ser Leu Asp Phe Glu Ile Arg Thr Gln Le - #u Ala Arg Glu Cys Ile         #     60                                                                      - Asn Arg Val Cys Glu Ala Ala Gly Leu Lys Se - #r Ala Gly Lys Arg Arg         # 80                                                                          - Leu Thr Asn Phe Ile Ser Asp Arg Pro Ser Me - #t Gln His Ala Gly Thr         #                 95                                                          - Asn Ile Ile Ile Asn Val Ser Ser Arg Ala Le - #u Ser Leu Ser Asn Val         #           110                                                               - Glu Thr Gly Glu Val Ile Ala Asn His Asn Me - #t Pro Arg Ile Ser Phe         #       125                                                                   - Ala Ser Gly Gly Asp Asn Asp Thr Leu Asp Ph - #e Leu Ala Tyr Ile Ala         #   140                                                                       - Lys Asn Glu Asp Glu Trp Arg Ala Cys Tyr Va - #l Leu Glu Cys Ala Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gly Gln Ser Glu Asp Leu Ile Val Thr Ile Gl - #y Lys Ala Phe Ala Leu         #               175                                                           - Arg Phe Asn Ala Leu Ser Arg Leu Asn Asp Pr - #o Ser Ala Asp Cys Asn         #           190                                                               - Ile Asn Gln Ser Cys Lys Glu Asn Val Lys Gl - #u Tyr Tyr Asn Asp Leu         #       205                                                                   - Pro Asn Lys Leu Pro Pro Glu Val Pro Glu Pr - #o Gln Gln Gln Gln Val         #   220                                                                       - Gln Gln Pro Leu His Pro His Ala Pro Arg Va - #l Ala Gln Leu Asn Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Lys Lys Pro Arg Asp Arg Leu Ser Ser Asn Le - #u Ile Asp Leu Asn Ser         #               255                                                           - Pro Pro Pro                                                                 __________________________________________________________________________

We claim:
 1. A peptide of the formula Ia

    X.sup.1 -A.sup.1 -A.sup.2 -X.sup.2 -Asn-X.sup.3 -X.sup.4 -P.Tyr-X.sup.5 -X.sup.6 -X.sup.7                                         Ia

wherein X¹ represents Lys, Arg, His, X² represents Glu, Asn, Tyr, Thr,Ser, X³ represents Pro, Met, Trp, Phe, Ala, Val, Leu, Ile, Gly, Cys, X⁴represents Gln, Asp, Asn, Tyr, Thr, Ser, X⁵ represents Phe, Trp, Pro,Leu, Ala, Val, Ile, Gly, Cys, Met, X⁶ represents Ser, Thr, Tyr, Asn,Glu, X⁷ represents Asp, Glu, and one of A¹ and A² represents Ile and theother of A¹ and A² represents Ile or Ala, which interferes with theinteraction of a PTB domain containing protein with a PTB domain bindingsite.
 2. A peptide of the formula Ia

    X.sup.1 -A.sup.1 -A.sup.2 -X.sup.2 -Asn-X.sup.3 -X.sup.4 -P.Tyr-X.sup.5 -X.sup.6 -X.sup.7                                         Ia

wherein X¹ represents Ser, Thr, Tyr, Asn or Glu, X² represents Glu, Asn,Tyr, Thr, Ser, X³ represents Pro, Met, Trp, Phe, Ala, Val, Leu, Ile,Gly, Cys, X⁴ represents Glu, Asp, X⁵ represents Phe, Trp, Pro, Leu, Ala,Val, Ile, Gly, Cys, Met X⁶ represents Ser, Thr, Tyr, Asn, Glu, X⁷represents Ala, Val, Leu, Ile, Gly, Cys, Phe, Trp, Met, Pro, and one ofA¹ and A² represent Ile and the other of A¹ and A² represents Ala, Val,Leu, Ile, Gly, Cys, Phe, Trp, Met or Pro, which interferes with theinteraction of a PTB domain containing protein with a PTB domain bindingsite.
 3. A peptide consisting of the sequenceHis-Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp;His-Ala-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp;His-Ile-Ala-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp;Tyr-Ala-Ile-Ser-Asn-Pro-Glu-P.Tyr-Leu-Ser-Ala;Thr-Trp-Ile-Glu-Asn-Lys-Leu-P.Tyr-Gly-Met-Ser-Asp;Thr-Trp-Ile-Glu-Asn-Lys-Leu-P.Tyr-Gly-Thr-Ser-Asp;Leu-Leu-Leu-Ser-Asn-Pro-Ala-P.Tyr.-Arg-Leu-Leu-Leu;Tyr-Ala-Ser-Ser-Asn-Pro-Glu-P.Tyr-Leu-Ser-Ala-Ser;Val-Ser-Val-Asp-Asn-Pro-Glu-P.Tyr-Leu-Leu-Asn-Ala;Ser-Leu-Leu-Ser-Asn-Pro-Thr-P.Tyr-Ser-Val-Met-Arg;Asn-Glu-Met-Ile-Asn-Pro-Asn-P.Tyr-Ile-Gly-Met-Gly;Glu-Met-Phe-Glu-Asn-Pro-Leu-P.Tyr-Gly-Ser-Val-Ser;Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe; Ala-Glu-Asn-Pro-Gln-P.Tyr-Phe;Ile-Ser-Asn-Pro-Glu-P.Tyr-Leu; orVal-Leu-Ala-Asp-Asn-Pro-Ala-P.Tyr-Arg-Ser-Ala (SEQ. ID. NOs. 1 to 3, 5to 14, 16, 18 and 19 in the Sequence Listing).
 4. A peptide consistingof the sequenceAla-Leu-Leu-Leu-Ser-Asn-Pro-Ala-P.Tyr.-Arg-Leu-Leu-Leu-Ala;Gly-Pro-Leu-Tyr-Ala-Ser-Ser-Asn-Pro-Glu-P.Tyr-Leu-Ser-Ala-Ser-Asp-Val-Phe;Pro-Val-Ser-Val-Asp-Asn-Pro-Glu-P.Tyr-Leu-Leu-Asn-Ala-Gln-Lys;Leu-Ser-Leu-Leu-Ser-Asn-Pro-Thr-P.Tyr-Ser-Val-Met-Arg-Ser-Lys;Val-Ser-Ser-Leu-Asn-Glu-Met-Ile-Asn-Pro-Asn-P.Tyr-Ile-Gly-Met-Gly-Pro-Phe;orLeu-Leu-Leu-Thr-Lys-Pro-Glu-Met-Phe-Glu-Asn-Pro-Leu-P.Tyr-Gly-Ser-Val-Ser-Ser-Phe(SEQ. ID. NOs. 20 to 25 in the Sequence Listing). 5.Cyclo-(Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Pro-Gly,cyclo-(Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp-Ala-Pro-Gly),cyclo-(His-Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp-Ala-Pro-Gly) orCys-Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Cys (SEQ. ID. NOs. 26 to
 29. 6. Amethod of treating a disorder mediated by the interaction of the PTBdomain, comprising the step of delivering to a patient a peptide of theformula Ia:

    X.sup.1 -A.sup.1 - A.sup.2 -X.sup.2 -Asn-X.sup.3 -X.sup.4 -P.Tyr-X.sup.5 -X.sup.6 -X.sup.7                                         Ia

wherein X¹ represents Lys, Arg, His, X² represents Glu, Asn, Tyr, Thr,Ser, X³ represents Pro, Met, Trp, Phe Ala, Val, Leu, Ile, Gly, Cys X⁴represents Gln, Asp, Asn, Tyr, Thr, Ser, X⁵ represents Phe, Trp, Pro,Leu, Ala, Val, Ile, Gly, Cys, Met, X⁶ represents Ser, Thr, Tyr, Asn,Glu, X⁷ represents Asp, Glu, and one of A¹ and A² represents Ile and theother of A¹ and A² represents Ile or Ala, which interferes with theinteraction of a PTB domain containing protein with a PTB domain bindingsite.
 7. A pharmaceutical composition for inhibiting the interaction ofa PTB domain with a phosphotyrosine-containing protein comprising apeptide and a pharmaceutically acceptable carrier, wherein the peptideis of the formula Ia:

    X.sup.1 -A.sup.1 -A.sup.2 -X.sup.2 -Asn-X.sup.3 -X.sup.4 -P.Tyr-X.sup.5 -X.sup.6 -X.sup.7                                         Ia

wherein X¹ represents Lys, Arg, His, X² represents Glu, Asn, Tyr, Thr,Ser, X³ represents Pro, Met, Trp, Phe, Ala, Val, Leu, Ile, Gly, Cys, X⁴represents Gln, Asp, Asn, Tyr, Thr, Ser, X⁵ represents Phe, Trp, Pro,Leu, Ala, Val, Ile, Gly, Cys, Met, X⁶ represents Ser, Thr, Tyr, Asn,Glu, X⁷ represents Asp, Glu, and one of A¹ and A² represents Ile and theother of A¹ and A² represents Ile or Ala, which interferes with theinteraction of a PTB domain containing protein with a PTB domain bindingsite.
 8. A peptide of the formula Ia as claimed in claim 1 wherein X¹represents His.
 9. A peptide of the formula Ia as claimed in claim 1wherein X² represents Glu.
 10. A peptide of the formula Ia as claimed inclaim 1 wherein X³ represents Pro.
 11. A peptide of the formula Ia asclaimed in claim 1 wherein X⁴ represents Gln.
 12. A peptide of theformula Ia as claimed in claim 1 wherein X⁵ represents Phe.
 13. Apeptide of the formula Ia as claimed in claim 1 wherein X⁶ representsSer.
 14. A peptide of the formula Ia as claimed in claim 1 wherein X⁷represents Asp.
 15. A peptide of the formula Ia:

    X.sup.1 -Ala-Ile-X.sup.2 -Asn-X.sup.3 -X.sup.4 -P.Tyr-X.sup.5 -X.sup.6 -X.sup.7

wherein X¹ represents Lys, Arg, His, X² represents Glu, Asn, Tyr, Thr,Ser, X³ represents Pro, Met, Trp, Phe, Ala, Val, Leu, Ile, Gly, Cys, X⁴represents Gln, Asp, Asn, Tyr, Thr, Ser, X⁵ represents Phe, Trp, Pro,Leu, Ala, Val, Ile, Gly, Cys, Met, X⁶ represents Ser, Thr, Tyr, Asn,Glu, X⁷ represents Asp, Glu, which interferes with the interaction of aPTB domain containing protein with a PTB domain binding site.
 16. Apeptide of the formula Ia as claimed in claim 1 wherein A² representsIle.
 17. A peptide of the formula Ia as claimed in claim 2 wherein X¹represents Tyr.
 18. A peptide of the formula Ia as claimed in claim 2wherein X² represents Ser.
 19. A peptide of the formula Ia as claimed inclaim 2 wherein X³ represents Pro.
 20. A peptide of the formula Ia asclaimed in claim 2 wherein X⁴ represents Glu.
 21. A peptide of theformula Ia as claimed in claim 2 wherein X⁵ represents Leu.
 22. Apeptide of the formula Ia as claimed in claim 2 wherein X⁶ representsSer.
 23. A peptide of the formula Ia as claimed in claim 2 wherein X⁷represents Ala.
 24. A method of treating a disorder mediated by theinteraction of the PTB domain, comprising the step of delivering to apatient a peptide of the formula Ia as claimed in claim
 2. 25. Apharmaceutical composition for inhibiting the interaction of a PTBdomain with a phosphotyrosine-containing protein comprising a peptide asclaimed in claim 2 and a pharmaceutically acceptable carrier.
 26. Apeptide of the formula Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Pro-Gly, SEQ IDNO:17.